WO2025101377A1

Movatterモバイル変換

Info

Publication number: WO2025101377A1
Application number: PCT/US2024/053054
Authority: WO
Inventors: Justin COSTA; Paulius Mielinis; Marco Serra
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2023-11-09
Filing date: 2024-10-25
Publication date: 2025-05-15
Anticipated expiration: 2026-05-09

Abstract

The present disclosure relates in some aspects to methods and compositions for matrix-assisted spatial analysis, where a biological sample is embedded in a matrix (e.g., a hydrogel matrix) and a target nucleic acid from the biological sample is tethered to the matrix, followed by removing the biological sample (e.g., by clearing the sample) from the matrix while the target nucleic acid remains tethered to the matrix. After sample removal, one or more probes can be hybrized to the tethered target nucleic acid. The target nucleic acid, the one or more probes, a product of the target nucleic acid, and/or a product of the one or more probes can be released from the matrix for capturing on an array, spatial barcoding on the array, and subsequent sequence analysis of spatially barcoded molecules, for example, using a next-generation sequencing method.

Description

(I), or a salt thereof, wherein each R^RNA is independently a reactive moiety capable of reacting with at least one 5’-phosphate group of the RNA or 5’- phosphate group of the RNA modified with a leaving group; each R^AM is independently an^ny-2785575 202412020440 attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent; L is a bond or a linker moiety; m is an integer from 1 to 4; and p is an integer from 1 to 4. [0273] In some embodiments, R^RNA, R^AM, L, m, and p are each as defined herein. It should be understood that every description, variation, embodiment or aspect of a moiety may be combined with every description, variation, embodiment or aspect of other moieties the same as if each and every combination of descriptions is specifically and individually listed. For example, every description, variation, embodiment or aspect provided herein with respect to R^RNA of Formula (I) may be combined with every description, variation, embodiment or aspect of L of Formula (I) the same as if each and every combination were specifically and individually listed. For another example, every description, variation, embodiment or aspect provided herein with respect to R^AM of Formula (I) may be combined with every description, variation, embodiment or aspect of L of Formula (I) the same as if each and every combination were specifically and individually listed. (i) Reactive moiety R^RNA [0274] In some embodiments, the attachment agent comprises a reactive moiety. As provided herein, the reactive moiety capable of covalently bonding to the ribonucleic acid can be any reactive moiety that reacts with and covalently bonds to a 5’-phosphate or a 5’-phosphate modified with a leaving group. In some variations, such reactive moiety capable of covalently bonding to the ribonucleic acid by covalently bonding to a 5’-phosphate on the ribonucleic acid. In some variations, such reactive moiety capable of covalently bonding to the ribonucleic acid by covalently bonding to a 5’-phosphate on the ribonucleic acid modified with a leaving group. [0275] In some embodiments, at least one reactive moiety is capable of reacting with at least one 5’-phosphate group of the RNA via an enzymatic reaction. In some embodiments, at least one reactive moiety of the attachment agent comprises or is a nucleic acid oligonucleotide comprising between 2 to 30 (e.g., between any of 2 to 25, 2 to 20, 2 to 15, or 5 to 15) nucleotide residues. In some embodiments, the nucleic acid oligonucleotide is a DNA oligonucleotide. The nucleic acid oligonucleotide can comprise any nucleic acid sequence, (e.g., any sequence of nucleotide residues). In some embodiments, the nucleic acid oligonucleotide comprises a random sequence. In some embodiments, the nucleic acid oligonucleotide comprises at least one thymine (T). In some embodiments, the nucleic acid oligonucleotide comprises a sequence of at least 2, 3, 4, 5, 6, 7, 8, or more thymines. In some embodiments, the nucleic acid oligonucleotide does not^ny-2785575 202412020440 comprise a sequence of thymines. In some embodiments, p is 4. In some embodiments, p is 3. In some embodiments, p is 2. In some embodiments, p is 1. In some embodiments, the attachment agent is of Formula (I-a):

wherein DNA-1 comprises a nucleic acid sequence of 7-15 nucleotide residues; each R^AM is independently an attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent; L is a bond or a linker moiety; and m is an integer from 1 to 4. [0276] In some embodiments, the DNA-1 comprises at least one thymine (T). In some embodiments, tethering RNA to the matrix comprises forming a covalent bond between a 3’-OH of the DNA-1 and the 5’-phosphate group of the RNA under the catalysis of a ligase. In some embodiments, the ligase is an RNA ligase. In some embodiments, the ligase is T4 RNA Ligase 1. [0277] In some embodiments, provided herein is a method comprising reacting a reactive moiety R^RNA with an RNA comprising a 5’-phosphate through an enzymatic process. In some embodiments, the attachment moiety R^AM is an acrydite. In some embodiments, the reactive moiety R^RNA is a nucleic acid, such as a DNA. In some embodiments, the DNA is a sequence of thymine residues. However, the nucleic acid oligonucleotide (e.g., DNA oligonucleotide) can comprise any sequence. In some embodiments, the nucleic acid oligonucleotide does not comprise a sequence of thymines. In some embodiments, the nucleic acid oligonucleotide comprises or is a random sequence. In some embodiments, the reactive moiety R^RNA (e.g., a nucleic acid comprising a 3’ hydroxyl group) reacts with an RNA comprising a 5’-phosphate under the catalysis of an RNA ligase, such as T4 RNA Ligase 1. In the example illustrated, R^RNA is a nucleic acid comprising 8 thymine (T). [0278] In some embodiments, at least one reactive moiety is capable of reacting with at least one 5’-phosphate group or 5’-phosphate group modified with a leaving group of the RNA via a non-enzymatic reaction. In some embodiments, at least one reactive moiety is capable of^ny-2785575 202412020440 reacting with a 5’-phosphate group modified with a leaving group of the RNA via a non- enzymatic reaction, such a substitution reaction. [0279] In some embodiments, at least one reactive moiety comprises or is a nucleophilic group capable of reacting with 5’-phosphate group modified with a leaving group of the ribonucleic acid. In some embodiments, at least one reactive moiety comprises or is an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, an ylide moiety, a hydrazide, a hydroxylamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, or an arylhydrazide, or any combination thereof. In some embodiments, at least one reactive moiety is or comprises an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, or an ylide moiety. [0280] In some embodiments, at least one reactive moiety is or comprises an amine moiety (e.g., -NHR or -NH₂), an alcohol moiety, or a thiol moiety that is capable of reacting with a 5’-phosphate group modified with a leaving group. In some embodiments, the reactive moiety of the attachment agent is or comprises an amine moiety (e.g., -NHR or -NH2). In some embodiments, the reaction of an amine moiety with a 5’-phosphate group modified with a leaving group of the ribonucleic acid forms a P-NH or -P-NR bond. In some embodiments, the reactive moiety of the attachment agent is or comprises an alcohol moiety (e.g., -OH). In some embodiments, the reaction of an alcohol moiety with a 5’-phosphate group modified with a leaving group of the ribonucleic acid forms a P-O bond. In some embodiments, the reactive moiety of the attachment agent is or comprises a thiol moiety (e.g., -SH). In some embodiments, the reaction of a thiol moiety with the 5’-phosphate group modified with a leaving group of the ribonucleic acid forms a P-S bond. [0281] In some embodiments, the method comprises anchoring both a 5’ end and a 3’ end of an RNA to the biological sample or to a matrix embedding the biological sample. In some instances, the method comprises anchoring a 5’ end of an RNA to the biological sample or to a matrix embedding the biological sample according to any of the embodiments described herein, and further comprises anchoring the 3’ end of the RNA to the matrix. In some embodiments, anchoring the 3’ end of the RNA to the matrix comprises contacting the biological sample with a probe that hybridizes to the 3’ end of the RNA (e.g., a probe comprising a plurality of thymine bases that hybridizes to a polyA tail of the RNA). In some embodiments, anchoring the 3’ end of the RNA comprises contacting the biological sample comprising the RNA with a formylation^ny-2785575 202412020440 reagent, wherein the RNA comprises a 2’,3’-vicinal diol and the formylation reagent converts the 2’,3’-vicinal diol moiety into a 2’,3’-dialdehyde moiety; and contacting the biological sample with a 3’-end attachment agent comprising at least one aldehyde-reactive group capable of reacting with at least one aldehyde of the 2’,3’-dialdehyde moiety of the ribonucleic acid to form a covalent bond and an attachment moiety capable of attaching covalently or non-covalently to an exogenous or endogenous molecule in the biological sample (e.g., to a matrix). [0282] In some embodiments, the 3’-end attachment agent comprises an aldehyde- reactive group. In some embodiments, the aldehyde-reactive group comprises or is a nucleophilic group capable of reacting with at least one aldehyde of the 2’,3’- dialdehyde moiety of the ribonucleic acid. In some embodiments, the reactive group comprises or is an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, an ylide moiety, a hydrazide, a hydroxylamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, or an arylhydrazide, or any combination thereof. In some embodiments, the aldehyde-reactive group or first reactive group of the attachment agent is or comprises an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, or an ylide moiety. In some embodiments, the aldehyde- reactive group of the 3’-end attachment agent is or comprises an amine moiety (e.g., -NHR or - NR2). In some embodiments, the reaction of an amine moiety with an aldehyde moiety of the ribonucleic acid forms an imine or an enamine. In some embodiments, the method comprises reduction of the imine (e.g., with NaBH₄, optionally with 0.2M NaBH₄). [0283] In some instances, the biological sample is contacted with a TN4 PNK to polish the 3' ends into diols and add 5' phosphates to the RNAs. In some embodiments, after T4 PNK polishing, the method comprises incubating the biological sample with a ligase and an attachment agent according to any of the embodiments of Formula (I-a) described herein (e.g., a tethering oligonucleotide comprising a 5’ acrydite). In some embodiments, the biological sample is contacted with between about 10 nM and about 100 nM of the tethering oligonucleotide. In some embodiments, the biological sample is contacted with about 20 nM of tethering oligonucleotide. In some embodiments, the method comprises incubating the biological sample with the tethering oligonucleotide, ligase, and a PEG reagent (e.g., PEG8000) at a temperature of about 25°C for about 1 hour to about 4 hours (e.g., at least about 1, 1.5, or 2 hours). In some embodiments, the method further comprises tethering the polished 3’ end of the RNA by RNA oxidation (e.g., with NaIO₄, optionally with 20mM NaIO₄), aldehyde coupling (e.g., with 2AEM,^ny-2785575 202412020440 methacrylamide, Et3N, and aniline), and imine reduction (e.g., with NaBH4, optionally with 0.2M NaBH₄). [0284] In some embodiments, the 5’-phosphate group modified with a leaving group are contacted with an attachment agent comprising an amine moiety (e.g., -NH2), an alcohol moiety, or a thiol moiety, to form a link between the attachment agent and the RNA. [0285] In some embodiments, the resulting 5’-phosphate group is further modified with a leaving group, and thereby forming an RNA comprising a 5’-phosphate group modified with a leaving group. In some embodiments, the modification comprise replacing one -OH group in the 5’-phosphate group with a leaving group. In some embodiments, the conjugation acid of the leaving group has a pKa of less than 8, such as less than about any of 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3. In some embodiments, the leaving group is any one of Cl, Br, I, -OR, -OC(O)R, -OS(O)₂R, or -NR¹R², wherein each R is independently haloalkyl, phenyl substituted with one or more alkyl or haloalkyl, or heteroaryl substituted with one or more alkyl or haloalkyl, and wherein R¹ is independently H, alkyl, or haloalkyl, R² is independently haloalkyl, phenyl substituted with one or more alkyl or haloalkyl, or heteroaryl substituted with one or more alkyl or haloalkyl, or R¹ and R² are taken together the N atom to which they are attached to form a heteroaryl. In some embodiments, the leaving group

wherein the wavy line denotes the attachment of the leaving group to the 5’ phosphate of the RNA. [0286] In some embodiments, the step of contacting the biological sample comprising an RNA and attachment agent further comprises contacting the biological sample with one or more reagents or under suitable conditions to facilitate the formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent. For example, in some embodiments, the methods provided herein comprise contacting the attachment agent and the biological sample with aniline and/or triethylamine to facilitate formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent. In some embodiments, the methods comprise contacting the attachment agent and the biological sample with triethylamine. In some embodiments, the methods comprise contacting the^ny-2785575 202412020440 attachment agent and the biological sample with triethylamine. In some embodiments, the methods do not comprise contacting the attachment agent and the biological sample with triethylamine to facilitate formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent. In some embodiments, the formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and optionally the reactive moiety of the attachment agent is further facilitated by an inorganic salt. In some embodiments, the inorganic salt is MgCl₂. In some embodiments, the formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent is facilitated by shifting the equilibrium to the right, optionally by removing the products after the substitution reaction. For example, in some embodiments when the leaving group is

the reaction is facilitated by selectively removing 1H-imidazole from the reaction system. (ii) Boronic Acid Moiety [0287] In some embodiments, RNA tethering to the matrix uses an attachment agent comprising boronic acid moieties in the matrix to passively capture 3’ RNA ends that have been polished, where the tethering occurs when the pH is greater than the pKa of the boronic acid moiety (which induces the formation of boronate esters with the 3’ RNA diols) and the tethering is reversed when the pH is below than the pKa. [0288] In some embodiments, a matrix (e.g., a hydrogel) and/or a matrix-forming agent comprising a boronic acid moiety can be used to tether an RNA of a sample (e.g., a cell or tissue sample embedded in the hydrogel) for subsequent removal of proteins (e.g., ribosome components) and lipids. In some embodiments, the hydrogel is sandwiched into a non-boronic acid hydrogel holding an array. The array embedded in the hydrogel can be generated by inverting an array in a 5’ up configuration into a matrix (e.g., a non-boronic acid hydrogel which can be non-reversible), such that the inverted array is in a 3’ up configuration and is embedded in the hydrogel. The inversion can help remove incorrectly synthesized molecules and introduce a more enzymatically friendly environment for downstream analysis, for instance, when a sample may need to interface with the oligonucleotides (e.g., capture probes) on the array. The oligonucleotides (e.g., capture probes) can be provided in a pattern on the array substrate, such as 81^ny-2785575 202412020440 in array spots. A drop in pH below the pKa of the boronic acid moieties in the tethered sample can release the RNAs on command to begin interfacing with the array oligonucleotides on the underlying hydrogel. In some embodiments, the tethered RNA is pressed against the array in the presence of a slightly acidic buffer to un-tether the RNA, where the proximity of the array oligonucleotide helps limit lateral diffusion through the hydrogels to mitigate mis-localization. In some embodiments, capacitance can be used to drive migration of the newly liberated RNA towards the array. For instance, thin copper plates connected to a power source can leverage capacitance to drive migration of the newly untethered RNA towards the oligonucleotides on the array. As capacitance is a function of the area between the two plates and the distance between them, the RNA migration can be tuned to add a degree of control over the un-tethered RNA. In some embodiments, a conductive coating (e.g., Indium Tin Oxide) on both sides of the sandwich can be used to permit electrophoresis of the RNA onto the array. For instance, the inverted array (in a 3’ up configuration) and the boronic acid hydrogel can be deposited on a methacrylic acid coated oxide surface, including but not limited to silicon oxide or quartz. A conductive coating that conducts current can be used to actively electrophorese the untethered RNA onto the array, providing control of RNA migration and/or capture using voltage, amperage, ionic conditions in the buffer, and/or temperature. [0289] In some embodiments, a boronic acid-based hydrogel can be used as a sample hydrogel for embedding a sample and a standard hydrogel can be used as an array hydrogel. In some embodiments, boronic acids may be embedded within the sample hydrogel matrix (e.g., a hydrogel mesh), to passively capture 3’ RNA ends that have been polished. In some approaches, the tethering of the RNA target molecules occurs if the pH of the system is greater than the pKa of the boronic acid. In some embodiments, this may be due to the formation of boronate esters between the boronic acid groups of the sample hydrogel with the 3’ RNA diols of the target RNA molecule. For embodiments, where the pH is below the pKa tethering may be revered and the tethered RNA target molecule can be released. In such an embodiment, no separate linker molecule is required. [0290] In some embodiments, two different hydrogels may be prepared: a boronic acid hydrogel (e.g., sample hydrogel) and a standard hydrogel (e.g., non-reversible hydrogel, non-boronic acid hydrogel, array hydrogel, or reader hydrogel). In some embodiments, capture probes (e.g., capture agents) of a spatial array are intermeshed in the standard hydrogel. The^ny-2785575 202412020440 sandwich arrangement, in accordance with some embodiments may include the boronic acid hydrogel-based reversible tethering scheme as depicted in FIG. 20A. [0291] In some embodiments, the boronic acid hydrogel may be formed over the tissue, where the RNA is tethered to the hydrogel. In some embodiments, a clearing step may be used to remove the ribosomes and/or prepare the sample to interface with the (non-boronic acid) hydrogel containing a spatial array for capturing nucleic acid molecules from the sample hydrogel. [0292] In some embodiments, the sample hydrogel may be sandwiched into the standard hydrogel. In some embodiments, the tethered RNA may be pressed against the capture array in the presence of a slightly acidic buffer, allowing the target RNA to be released from the sample hydrogel. In some embodiments, due to the proximity of the capture probes (e.g., array oligos), lateral diffusion may be negligible as the target RNA diffuses through both the sample hydrogel and the capture hydrogel, as depicted in FIG. 20A, thereby mitigating mis-localization. [0293] In some examples, pH triggered release of target molecules (e.g., target nucleic acid, target RNA, target RNA molecules) tethered to a hydrogel may be followed by diffusion of the mRNA target to the capture array. In some examples, capacitance may be utilized to limit diffusion in one direction (e.g., anisotropic diffusion), where released target molecules are directed toward the capture array, thereby increasing the efficiency of the capture process. In some embodiments, an external field driving the electrophoretic migration of the target molecule to the capture array (e.g., hydrogel barcoded capture array) is provided by thin copper plates connected to a power source as depicted in FIG. 20B where the copper plates act as or are connected to a cathode and an anode, respectively. The capacitance, C, of a pair of plates of a given material can be described as a function of three parameters: the plate area, A; the distance, d, between the two plates and the dielectric constant, ed, of the material from which the plates are made as shown in Function 1: C = ede0A/2d. [0294] In some embodiments, use of thin conductive layers applied to both the sample substrate and the capture array substrate may provide a capacitance allowing for electrophoretic migration as described in the current application. FIG. 20C depicts a thin film of indium tin oxide (ITO) deposited on both the sample substrate and the capture array substrate in accordance with some embodiments. In some embodiments, the spatial array hydrogel and the^ny-2785575 202412020440 boronic acid hydrogel are deposited on a methacrylic acid coated, conductive oxide surface. In some embodiments, the oxide surface may comprise ITO. In some embodiments, the conductive oxide surface may be used provide a capacitive force allow for electrophoretic migration of released target molecules the hydrogel barcoded capture array (e.g., ITO coated substrate with capture probes). ITO coated substrates may allow for a great degree of control in the release and electrophoretic driven migration (e.g., directionally directed, or anisotropic diffusion), as voltage, amperage, ionic conditions in the buffer and temperature may be optimized and controlled. (iii) Attachment Moiety [0295] In some embodiments, the attachment agent comprises an attachment moiety. In some embodiments, an attachment moiety is any functional group that interacts with a matrix- forming agent and, in some embodiments, the attachment moiety comprises or is a group capable of reacting with, covalently binding, or non-covalently binding to a complementary reactive group on the matrix-forming agent. [0296] In some embodiments, at least one attachment moiety is capable of attaching covalently to a matrix-forming agent. In some embodiments, at least one attachment moiety is or comprises an alkenyl, alkynyl, allyl or vinyl moiety, ally ester moiety, an acrylamide moiety, an amide moiety, an alcohol moiety, a polyol moiety, a furan moiety, a maleimide moiety, a norbornene moiety, a thiol moiety, a sulfide moiety, a phenol moiety, a urethane moiety, a cyano moiety, an amino moiety, an isocyanate moiety, an isothiocyanate moiety, an ether moiety, a dextran moiety, or an alginate moiety. [0297] In some embodiments, at least one attachment moiety comprises or is an electrophilic group that is capable of interacting with a reactive nucleophilic group present on the matrix-forming agent to provide a covalent bond between the attachment moiety and the matrix- forming agent. In some embodiments, the nucleophilic groups on the matrix-forming agent having that capability include but are not limited to, sulfhydryl, hydroxyl and amino functional groups. In some embodiments, at least one attachment moiety comprises or is a maleimide, haloacetamide, or NHS ester. [0298] In some embodiments, at least one attachment moiety comprises or is a nucleophilic group that is capable of interacting with a reactive electrophilic group present on the matrix-forming agent to provide a covalent bond between the attachment moiety and the matrix- forming agent. In some embodiments, at least one attachment moiety comprises or is a thiol,^ny-2785575 202412020440 phenol, amino, hydrazide, hydroxylamine, hydrazine, thiosemicarbazone, hydrazine carboxylate, or arylhydrazide. In some embodiments, each attachment moiety is independently or independently comprises a phenol moiety, an alkyne moiety, a norbornene moiety, a sulfide moiety, a furan moiety, a maleimide moiety, or an allyl ester moiety. [0299] In some embodiments, at least one attachment moiety comprises or is a click functional group. Suitable click functional groups include functional groups compatible with a nucleophilic addition reaction, a cyclopropane-tetrazine reaction, a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, an alkyne hydrothiolation reaction, an alkene hydrothiolation reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron- demand Diels-Alder (IED-DA) reaction, a cyanobenzothiazole condensation reaction, an aldehyde/ketone condensation reaction, and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, the attachment moiety(ies) or second reactive group comprise or is any functional group involved in click reactions. In some embodiments, such click reactions involve (i) azido and cyclooctynyl; (ii) azido and alkynyl; (iii) tetrazine and dienophile; (iv) thiol and alkynyl; (v) cyano and amino thiol; (vi) nitrone and cyclooctynyl; or (vii) cyclooctynyl and nitrone. It should be recognized that in instances in which the attachment moiety comprises or is a click functional group, the matrix-forming agent to which it is capable of forming a covalent bond comprises the complementary click functional group to that of the attachment moiety. For example, in some embodiments, the attachment moiety comprises or is an azide moiety and the matrix-forming agent comprises a complementary alkyne moiety, or vice versa. [0300] In some embodiments, at least one attachment moiety comprises or is a group capable of reacting with a matrix-forming agent. As detailed herein, examples of matrix-forming agents include but are not limited to acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof. In some embodiments, at least one^ny-2785575 202412020440 attachment moiety comprises or is an alkenyl, allyl or vinyl moiety, an amide moiety, an alcohol moiety, a polyol moiety, a furan moiety, a maleimide moiety, a norbornene moiety, a thiol moiety, a phenol moiety, a urethane moiety, a cyano moiety, an isocyanate moiety, an isothiocyanate moiety, an ether moiety, a dextran moiety, or an alginate moiety. In some embodiments, at least one attachment moiety comprises or is an alkenyl, allyl or vinyl moiety (e.g., -C=C- or HC=C- or HC=C-CH2-), such as in N-(2-aminoethyl)methacrylamide, 2- aminoethyl methacrylate, 2-aminoethyl (E)-but-2-enoate, 2-aminoethyl methacrylate or methylacrylamide, or norbornene. Such alkenyl, allyl or vinyl moieties may be suitable for reaction with matrix-forming agents. [0301] In some embodiments, at least one attachment moiety comprises or is an acrylate moiety, methacrylate moiety, acrylamide moiety, methacrylamide moiety, biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety. In certain embodiments, at least one attachment moiety comprises or is a biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety. [0302] In some embodiments, the attachment agent is of Formula (I-b):

wherein R^RNA is any of the reactive moiety provided herein; wherein L is a bond or any of the linker moiety provided herein; wherein W is independently H or C_1-6 alkyl; wherein Y is H or C1-6 alkyl; and wherein X is NH, N(C_1-6 alkyl), or O. [0303] In some embodiments, the attachment agent is of Formula (I-c):

wherein n is an integer from 0 to 50; wherein Z is CH₂ or O;^ny-2785575 202412020440 m is an integer from 1 to 4; p is an integer from 1 to 4; wherein R^RNA is any of the reactive moiety provided herein; and wherein R^AM is phenol, azide, alkyne, norbornene, sulfide, furan, maleimide, or allyl ester. [0304] In some embodiments, the formation of a covalent or non-covalent bond between the attachment moiety and the matrix-forming agent is mediated by an external reagent or stimulus. For example, in some embodiments, the formation of a covalent or non-covalent bond between the attachment moiety and the matrix-forming agent is initiated or induced by an enzyme, a catalyst, chemical reagents (e.g., acid, base, reducing agent, oxidant, etc.), heat, and/or light. In some embodiments, a covalent bond is formed between the attachment moiety and the matrix-forming agent. In some embodiments, the step of contacting the biological sample and attachment agent further comprises contacting the biological sample with one or more reagents or under suitable conditions to facilitate the formation of a covalent bond between at least one attachment moiety of the attachment agent and the matrix-forming agent. For example, in some embodiments wherein the attachment moiety comprises an alkene or a click functional group, the method optionally further comprises adding reagents to activate the alkene or click functional group, such as a radical initiator or a copper catalyst, respectively. In other embodiments wherein at least one attachment moiety comprises an alkenyl, allyl or vinyl moiety, the method optionally further comprises exposing the biological sample and attachment agent to (ultraviolet) light or heat to facilitate formation of a covalent bond. In some embodiments wherein at least one attachment moiety comprises or is a norbornene moiety, furan moiety, maleimide moiety, or other alkenyl, allyl or vinyl moiety, the method optionally further comprises exposing the sample to light or heat. In yet other embodiments, the method may further comprise adding an enzyme to facilitate formation of a covalent bond. For example, in some embodiments wherein at least one attachment moiety comprises or is a phenol moiety, the method optionally further comprises adding horseradish peroxidase (HRP). [0305] In some embodiments, the attachment moiety is capable of attaching non- covalently to a matrix-forming agent. In some embodiments, the attachment moiety is capable of attaching non-covalently to an exogenous molecule in the biological sample. In some embodiments, the attachment moiety is capable of attaching non-covalently to an endogenous molecule in the biological sample. In some embodiments, the attachment moiety comprises or is^ny-2785575 202412020440 a group capable of binding to a matrix-forming agent via non-covalent interaction, such as but not limited to hydrogen bonding, van der Waals interaction, and/or pi-stacking. [0306] In some embodiments, the attachment agent is biotinylated. In some embodiments, the attachment moiety is a biotin moiety or a derivative thereof. (iv) Linker L [0307] In some embodiments, the attachment agent comprises a linker and/or linker moiety. In some embodiments, such as some embodiments of Formula (I), (I-a), or (I-b), L is a bond. In some embodiments of Formula (I), L is a linker moiety. In some embodiment, L comprises or is an unbranched or branched C₁-C₁₅₀ alkylene, which can be interrupted by 1 to 50 independently selected O, NH, N, S, C6-C12 arylene, or 5- to 12-membered heteroarylene. In some embodiments, L comprises or is an unbranched and uninterrupted C1-C150 alkylene. In some embodiments, L comprises or is a branched and uninterrupted C₁-C₁₅₀ alkylene. In some embodiments, L comprises or is an unbranched C1-C150 alkylene interrupted by 1 to 50 NH, O, or S. In some embodiments, L comprises or is

, Z is CH₂, O, S; or NH; and n is an integer between 0 and 50. In some embodiments, L is

, Z is CH₂, O, S; or NH; and n is an integer between 0 and 50 (e.g., between 0 and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L comprises or is

, Z is CH₂, O, S; or NH; and n is an integer between 1 and 10. In some embodiment, L comprises or is

, Z is CH₂, O, S; or NH; and n is 6. In some embodiments, L comprises or is an unbranched C₁-C₁₅₀ alkylene interrupted by 1 to 50 oxygen. In some embodiments, L comprises a polyethylene glycol portion or is a polyethylene glycol moiety. In some embodiments, L comprises or is

, and n is an integer between 0 and 50. In some embodiments, L comprises or is

,^ny-2785575 202412020440 and n is an integer between 1 and 10. In some embodiment, L is

, and n is 6. In some embodiment, L is

, and n is an integer between 0 and 50 (e.g., between 0 and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L comprises an oligoethylene glycol. In some embodiments, L is an oligoethylene glycol moiety. In some embodiments, L comprises or is a branched C₁-C₁₅₀ alkylene interrupted by 1 to 50 oxygen. In some embodiments, L comprises or is an unbranched C1-C150 alkylene interrupted by 1 to 50 sulfurs. In some embodiments, L comprises or is

, and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L comprises or is a branched C1-C150 alkylene interrupted by 1 to 50 sulfurs. In some embodiments, L comprises or is a branched C1-C150 alkylene interrupted by 1 to 50 -NH-. In some embodiments, L comprises or is an unbranched C₁-C₁₅₀ alkylene interrupted by 1 to 50 - NH-. In some embodiments, L comprises or is

, and n is an integer between 0 and 50. In some embodiment, L is

, and n is 6. In some embodiment, L is

, and n is an integer between 0 and 50 (e.g., between 0 and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L is a branched C1-C150 alkylene interrupted by 1 to 50 -NH-, wherein the -NH- is not at a branching point. In some embodiments, L comprises or is a branched C1-C150 alkylene interrupted by 1 to 50 -N-, wherein the -N- is at a branching point. In some embodiments, L^ny-2785575 202412020440 comprises or is an unbranched or branched C1-C150 alkylene interrupted by 1 to 50 independently selected C₆-C₁₂ arylene, for example, any of phenyl or naphthalene. In some embodiments, L comprises or is an unbranched or branched C1-C150 alkylene interrupted by 1 to 50 independently selected 5- to 12-membered heteroarylene, for example, any of pyridine, furan, pyrrole, or thiophene. (v) Exemplary Formulae [0308] In some embodiments, the attachment agent as described herein (e.g., the attachment agent of Formula (I)) comprises any of 1, 2, 3, or 4 reactive groups (e.g., R^RNA). The reactive groups RRNA of Formula (I) are each independently selected and as defined in Section (B) (i) above. In some embodiments, the attachment agent is a bifunctional molecule comprising one reactive group. In some embodiments of Formula (I), p is any of 1, 2, 3, or 4. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one reactive groups R^RNA (e.g., p is any of 2, 3, or 4), wherein the R^RNA groups can be the same group, selected from the embodiments provided herein. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one reactive groups R^RNA (e.g., p is any of 2, 3, or 4), wherein each R^RNA is independently selected from the embodiments provided herein, provided the more than one reactive groups are chemically compatible and have chemically compatible ribonucleic- binding mechanisms or reactions. [0309] In some embodiments, the attachment agent (e.g., Formula (I)) comprises any of 1, 2, 3, or 4 attachment moieties (e.g., R^AM). The attachment moieties R^AM of Formula (I) are each independently selected and as defined in Section (B)(iii) above. [0310] In some embodiments, R^AM is capable of reacting with a matrix-forming agent to form a covalent bond. In some embodiments, R^AM is capable of reacting with a matrix- forming agent. In some embodiments, R^AM is In some embodiments, R^AM is an alkenyl, allyl or vinyl moiety, an amide moiety, an alcohol moiety, a polyol moiety, a furan moiety, a maleimide moiety, a norbornene moiety, a thiol moiety, a phenol moiety, a urethane moiety, a cyano moiety, an isocyanate moiety, an isothiocyanate moiety, an ether moiety, a dextran moiety, or an alginate moiety. In some embodiments, R^AM is a biotin moiety or a derivative thereof. In some embodiments, R^AM is an acrylate moiety, methacrylate moiety, acrylamide moiety, methacrylamide moiety, biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety. In certain^ny-2785575 202412020440 embodiments, R^AM is a biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety. [0311] In some embodiments of Formula (I), m is any of 1, 2, 3, or 4. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one attachment moieties R^AM (e.g., m is any of 2, 3, or 4), wherein the attachment moieties are the same group, selected from the embodiments provided herein. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one attachment moieties R^AM (e.g., m is any of 2, 3, or 4), wherein each R^AM is independently selected from the embodiments provided herein, provided the more than one reactive groups are chemically compatible and their binding mechanism or reactions to the matrix-forming agent are also chemically compatible. [0312] In some embodiments, the attachment agent is a compound of Formula (I-d),

or a salt thereof, wherein W is independently H or C1-6 alkyl (e.g., CH3); Y is H or C1-6 alkyl (e.g., CH3); X is NH, N(C_1-6 alkyl), or O; Z is CH2, O, or S; n is an integer from 0 to 50 (e.g., an integer between 1 and 20 or between 1 and 10); and DNA-1 comprises a nucleic acid sequence of 7-15 nucleotide residues. [0313] In some embodiments, the attachment agent is a compound of Formula (I-e),

or a salt thereof, wherein W is independently H or C_1-6 alkyl (e.g., CH₃); Y is H or C1-6 alkyl (e.g., CH3);^ny-2785575 202412020440 X is NH, N(C1-6 alkyl), or O; Z is CH₂, O, or S; n is an integer from 0 to 50 (e.g., an integer between 1 and 20 or between 1 and 10); and

[0314] In some embodiments, the attachment agent is of Formula (I-f):

wherein n is an integer from 0 to 50 (e.g., an integer between 1 and 20 or between 1 and 10); wherein Z is CH₂ or O; wherein R^RNA is NH2 or OH; and wherein R^AM is phenol, azide, alkyne, norbornene, sulfide, furan, maleimide, or allyl ester. [0315] In some embodiments of any of Formulae (I-c), (I-d), (I-e), and (I-f), L is , Z is O and n is 6. In some embodiments wherein L is

, L is a hexaethylene glycol moiety. C. Spatial Profiling of RNA [0316] In some embodiments, provided herein is a method for spatial analysis of biological molecules in a cell or tissue sample on a substrate (e.g., a slide or slip such as a glass or plastic slide or slip), comprising tethering the biological molecules in the cell or tissue sample to a matrix (e.g., a hydrogel), followed by releasing the tethered biological molecules for subsequent analysis. In some embodiments, the biological molecules comprise nucleic acid molecules (e.g., RNA such as mRNA). In some embodiments, the cell or tissue sample is a fresh frozen sample or a formalin-fixed paraffin-embedded sample, such as a fresh frozen tissue section or a formalin-fixed paraffin-embedded tissue section on a substrate. In some embodiments, the method comprises embedding the fresh frozen sample or the formalin-fixed paraffin-embedded sample on a sample substrate in a hydrogel, and tethering nucleic acid 92^ny-2785575 202412020440 molecules (e.g., RNA such as mRNA) in the sample to the hydrogel during or after the formation of the hydrogel embedding the sample on the sample substrate. In some embodiments, a formalin-fixed paraffin-embedded sample is decrosslinked prior to embedding the sample in the hydrogel, and the decrosslinking may allow more RNA molecules to interact with the hydrogel matrix for tethering (e.g., RNA molecules previously crosslinked with cellular components such as proteins or lipids may be decrosslinked such that they can be tethered to the hydrogel matrix). In some embodiments, a formalin-fixed paraffin-embedded sample is decrosslinked after embedding the sample in the hydrogel, and decrosslinking the hydrogel-embedded sample may decrosslink RNA from cellular components such as proteins or lipids while allowing the RNA to remain tethered (via the 3’ end or 5’ end) to the hydrogel. [0317] In some embodiments, at least one or more nucleic acid molecules (e.g., RNA such as mRNA) in the sample are 3’ tethered to the hydrogel. In some embodiments, at least one or more nucleic acid molecules (e.g., RNA such as mRNA) in the sample are 5’ tethered to the hydrogel. In some embodiments, the 3’ tethering and/or the 5’ tethering comprises use of an attachment agent. In some embodiments, the 3’ tethering and/or the 5’ tethering are reversed to allow release of the nucleic acid molecules (e.g., RNA such as mRNA) from the matrix on the sample substrate. In some embodiments, the released nucleic acid molecules (e.g., RNA such as mRNA) are captured by capture probes on an arrayed substrate of capture probes. In some embodiments, the sample substrate and the array substrate are the same substate. In some embodiments, the sample substrate and the array substrate are separate substates which can be brought into physical proximity to each other to facilitate the release, migration, and/or capture of the nucleic acid molecules. In some embodiments, a method disclosed herein is used for spatial transcriptomic profiling of an FF tissue section or an FFPE tissue section mediated by 3’ or 5’ target (e.g., nucleic acid molecules such as RNA, e.g., mRNA, or ligation products that serve as proxies of target RNA) tethering to a hydrogel. [0318] In some embodiments, provided herein is a method for spatial analysis of a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target nucleic acid is tethered to a matrix of the hydrogel; b) digesting and removing the embedded biological sample from the hydrogel; c) releasing the target nucleic acid from the matrix of the hydrogel; d) hybridizing the released target nucleic acid to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode^ny-2785575 202412020440 associated with the location of the capture probe on the array; and e) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, thereby analyzing the location of the target nucleic acid in the biological sample. In some embodiments, the target nucleic acid is a cellular nucleic acid, such as genomic DNA or mRNA. In some embodiments, the target nucleic acid is a product of a cellular nucleic acid (e.g., genomic DNA or mRNA), the product can be generated in situ at a location in the biological sample (e.g., prior to digesting and removing the embedded biological sample from the hydrogel), and the product can be generated before, during, or after embedding the biological sample in the hydrogel. In some embodiments, the product can be a cDNA of an RNA molecule in the biological sample. In some embodiments, the target nucleic acid is a nucleic acid probe that hybridizes to a cellular nucleic acid (e.g., genomic DNA or mRNA) or a nucleic acid probe that hybridizes to a product (e.g., cDNA) of the cellular nucleic acid. In some embodiments, the target nucleic acid is a product of nucleic acid probes that hybridize to a cellular nucleic acid (e.g., genomic DNA or mRNA) or a product of nucleic acid probes that hybridize to a product (e.g., cDNA) of the cellular nucleic acid. In some embodiments, the product of the nucleic acid probes is a ligation product, which can be analyzed as proxy of the cellular nucleic acid. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and a hydrogel on the substrate. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and no hydrogel on the substrate. [0319] In some embodiments, provided herein is a method for analyzing a biological sample comprising tethering a target RNA to a hydrogel matrix, clearing the biological sample, and releasing the target RNA from the hydrogel matrix. In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA from the biological sample is tethered to a matrix of the hydrogel; b) digesting and removing the embedded biological sample from the hydrogel; c) applying a first and a second probe to the hydrogel, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA and the second probe comprises a sequence substantially complementary to a second portion of the target RNA and a capture domain that is substantially complementary to a capture domain of a capture probe on an array, wherein the^ny-2785575 202412020440 capture probe further comprises a spatial barcode; d) hybridizing the first probe and the second probe to the target RNA tethered to the matrix of the hydrogel; e) ligating together the first probe and the second probe hybridized to the target RNA in the hydrogel, thereby generating a ligation product that is a proxy of the target RNA; f) releasing the ligation product from the target RNA in the hydrogel and hybridizing the released ligation product to the capture domain of the capture probe on the array; g) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the ligation product, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and a hydrogel on the substrate. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and no hydrogel on the substrate. [0320] Turning to FIG. 12A, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. an FFPE sample) with the steps of 1) baking the slide comprising the sample; 2) dewaxing the sample; 3) rehydrating the sample; 4) performing H&E staining; 5) imaging the sample (e.g. using brightfield (BF) imaging); and/or 6) destaining the sample (1201). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1202). In some embodiments, the method further comprises tethering a target (e.g., a target nucleic acid such as RNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1203). In some embodiments, the method further comprises decrosslinking the sample in the matrix (1204). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while tethered target remains attached to matrix (1205). In some embodiments, the method further comprises contacting the tissue sample with a probe or probe set that directly or indirectly binds to the target tethered to the matrix, followed by one or more optional wash(es) (1206). In some embodiments, the method further comprises ligating the probe or probe set bound to the target within the matrix (e.g., RNA-templated ligation), followed by one or more optional wash(es) (1207). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and a ligated probe is hybridized to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1208). In some embodiments, the method^ny-2785575 202412020440 further comprises releasing the target and/or the ligated probe (e.g., with RNase H treatment) for capture on the array (1209). In some embodiments, the method further comprises elongating the probe captured on array and proceeding with library preparation (1210). [0321] In some embodiments, provided herein is a method for analyzing a biological sample disclosed herein may comprise embedding the biological sample, such as an FFPE sample, in a hydrogel matrix, wherein a target RNA or a ligation product may be tethered to the hydrogel matrix. In some embodiments, the target RNA or ligation product may be tethered by 3’ or 5’ tethering to the hydrogel. In some embodiments, the method may comprise removing the embedded biological sample from the hydrogel matrix as depicted in FIG. 12A, leaving the target RNA or ligation product tethered to the hydrogel matrix. In some embodiments, the removing comprises clearing the sample embedded in the hydrogel. [0322] In some embodiments, the method as depicted in FIGS. 12A-12C may comprise applying a first probe and a second probe to the hydrogel matrix, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA. In some embodiments, the second probe may comprise a sequence substantially complementary to a second portion of the target RNA and a capture sequence configured to hybridize to or be ligated to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode. In some embodiments, the capture sequence in the second probe is substantially complementary to the capture domain in the capture probe. In some embodiments, the capture sequence comprises a sequence that is substantially complementary to a first portion of a splint oligonucleotide, which splint oligonucleotide further comprises a second portion that is substantially complementary to a sequence of the capture domain. As such, the first capture sequence and the capture domain, upon hybridization to the splint oligonucleotide, are configured to be ligated to each other, with or without gap filling prior to the ligation. [0323] Exemplary splint oligonucleotides and methods of using splint oligonucleotides for capturing target molecules (e.g., target RNA molecules, transposed DNA, and/or analyte capture agents for proteins described in Section II-C through Section II-F of the present disclosure) to capture probes on an array include but are not limited to those described in US 2023/0175045 A1 and US 2023/0287481 A1, which are incorporated herein by reference in their entireties.^ny-2785575 202412020440 [0324] In some embodiments, the probe with a sequence substantially complementary to the first portion of the target RNA is referred to as a left-hand side (e.g., LHS) probe as depicted in FIG.12B. In some embodiments, the functional sequence of the first probe may comprise a functional sequence (e.g., 5’ Read 2S) as depicted in FIG. 12B. In some embodiments, the probe with a sequence substantially complementary to a second portion of the target RNA is referred to as a right-hand side (e.g., RHS) probe. In some embodiments, the capture sequence of the second probe may comprise a poly(A) sequence. As depicted in FIG. 12B, in some embodiments, the method may comprise hybridizing the first probe and the second probe to the target RNA tethered to the hydrogel matrix. In some embodiments, the method may further comprise washing the hydrogel matrix containing the tethered target RNA and the first and second probes hybridized thereon (“washes in gel” in the probe hybridization step), for instance, to remove nonspecifically hybridized first and/second probes and/or to remove first and/second probes that contain one or more mismatches to the target RNA. In some embodiments, the method further comprises ligating together the first probe and the second probe hybridized to the target RNA tethered to the hydrogel matrix, thereby generating a ligation product that is a proxy of the target RNA. In some embodiments, the method further comprises washing the hydrogel matrix containing the tethered target RNA and the ligation product hybridized thereon (“washes in gel” in the probe ligation step), for instance, to remove first and/second probes that are not ligated to each other, and/or to remove ligation products that contain one or more mismatches which are less stably hybridized to the target RNA compared to ligation products containing no mismatch with the target RNA. [0325] In some embodiments, the first and second probes (e.g., the LHS and RHS probes shown in FIG. 12B) are provided in a probe mix which is applied to the hydrogel matrix, and the first and second probes are not immobilized to a substrate. In some embodiments, the first and second probes are dissolved in a solution such that the probes are free to move in the solution and are not immobilized to any location in the hydrogel or on the sample substrate until probe hybridization to the target RNA. [0326] In some embodiments, the method may comprise releasing the ligation product from the hydrogel matrix. In some embodiments, the ligation product is released from the target RNA. In some embodiments, the target RNA and/or a linkage between the target RNA and the matrix is cleaved to release the tethered target RNA or a portion thereof. In some^ny-2785575 202412020440 embodiments, the tethered target RNA is relased via cleavage of a linker between the target RNA and the hydrogel matrix. In some embodiments, the target RNA and the ligation product hybridized thereon are released together from the hydrogel matrix. In some embodiments, the ligation product is separated from the target RNA hybridized thereon, e.g., through dehybridization and/or RNA digestion, e.g., using an RNase H to cleave the phosphodiester bonds of RNA in a double-stranded RNA:DNA hybrid. [0327] In some embodiments, the method may comprise releasing the ligation product from the hydrogel matrix and hybridizing or ligating the released ligation product to the capture domain of the capture probe on a spatial array. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. In some embodiments, the method may comprise “sandwiching” (e.g., “Hydrogel/hydrogel” sandwich as depicted in FIG. 12A), where the RNA- tethered “tissue” hydrogel is aligned with a barcoded solid support (e.g., a spatial array such as a hydrogel barcoded capture array), for example, using a device described in any of FIGS. 1A-4B and described in Section I. In some embodiments, the method may comprise determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the ligation product or a complement thereof, and the sequence of the spatial barcode or a complement thereof. [0328] In some embodiments, the biological sample may be a fresh cell or tissue sample. In some embodiments, the biological sample may be a frozen cell or tissue sample. In some embodiments, the biological sample may be a fixed cell or tissue sample. In some embodiments, the biological sample may be an FFPE tissue section. In some embodiments, the biological sample may be a crosslinked biological sample, and the crosslinked biological sample may be embedded in the hydrogel matrix and the target RNA in the crosslinked biological sample may be tethered to the hydrogel matrix. In some embodiments, the method comprises decrosslinking the crosslinked biological sample (e.g., an FFPE sample that has been dewaxed, rehydrated, stained (e.g., using H&E stain), and/or destained) embedded in the hydrogel matrix. [0329] In some embodiments, the functional sequence in the first probe is in a 5’ overhang, which is not complementary to the target RNA, upon hybridization of the first probe to the target RNA. In some embodiments, the functional sequence in the first probe comprises a^ny-2785575 202412020440 primer binding sequence or a complement thereof. In some embodiments, the capture sequence in the second probe is in a 3’ overhang, which is not complementary to the target RNA, upon hybridization of the second probe to the target RNA. In some embodiments, the capture sequence in the second probe comprises a 3’ polyadenine sequence. In some embodiments, the first probe and the second probe are ligated using the target RNA as a template without gap filling prior to the ligation. In some embodiments, the first probe and the second probe are ligated with gap filling prior to the ligation. In some embodiments, the gap filling comprises hybridization of a gap-fill oligonucleotide to the target RNA and/or primer extension of the first probe or the second probe by a polymerase. In some embodiments, the gap filling comprises primer extension of the 3’end of the first probe by a polymerase with little or no strand displacement activity, thereby forming a nick between the 3’ end of the extended first probe and the 5’ end of the second probe, followed by RNA-templated ligation of the juxtaposed ends. In some embodiments, the gap filling comprises extension via a polymerase of the LHS 3’ probe end to meet the 5’ end of the RHS probe, followed by ligation. In some embodiments, hybridization of a third oligonucleotide in the gap between the first and the second probes followed by ligation of the third oligonucleotide to the LHS and the RHS probes. [0330] In some embodiments, the first probe and/or the second probe is comprised primarily of DNA and comprises one or more ribonucleotide residues. In some embodiments, the first probe and/or the second probe comprises no more than four consecutive ribonucleotide residues. In some embodiments, the one or more ribonucleotide residues are no more than five nucleotides from the 3’ or the 5’ end of the first probe and/or the second probe. In some embodiments, the first probe comprises a 3’ terminal ribonucleotide residue. In some embodiments, the second probe comprises a 5’ flap which is cleaved (e.g., by a flap endonuclease such as FEN1) prior to ligation of the cleaved second probe to the first probe. More disclosure on flap endonuclease usage can be found, for example, in US 2020/0224244, which is herein incorporated by reference in its entirety. [0331] In some embodiments, the releasing comprises releasing the tethered RNA from the hydrogel matrix. In some embodiments, the releasing comprises releasing the ligation product from the target RNA without cleaving the ligation product. In some embodiments, the releasing comprises contacting the hydrogel matrix with an RNase H.^ny-2785575 202412020440 [0332] In some embodiments, the capture domain in the capture probe comprises a 3’ poly(dT) sequence. In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising, from 5’ to 3’, a sequence of the released ligation product and a complementary sequence of the spatial barcode. For instance, as depicted in FIG. 12C, the spatially labeled polynucleotide can be generated by primer extension of the poly(A) hybridized to the poly(dT)VN using the capture probe as a template, and the spatially labeled polynucleotide comprises, from 5’ to 3’: a functional sequence (e.g., 5’ Read 2S), a sequence of the first probe, a sequence of the second probe, poly(A), a complement of the UMI, a complement of the spatial barcode, and a complement of a functional sequence in the capture probe (e.g., Read 1). In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released ligation product. [0333] In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the array for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the array may be analyzed by nucleic acid sequencing. In some embodiments, an amplification (e.g., a linear amplification) can be performed on-surface and post-extension to release a spatially labeled polynucleotides for sequencing analysis. In some embodiments, after extension of a captured ligation product of probes ligated using an RNA as a template, extended products (e.g., single copies) can be denatured and amplified, e.g., in a tube. In some embodiments, after extension of a captured ligation product of probes ligated using an RNA as a template, extended products can be treated with an enzyme (e.g., Exo I selectively catalyzes the degradation of single-stranded DNA in the 3′ to 5′ direction, whereas double-stranded DNA is protected from degradation) and amplified, e.g., using linear amplification after extension, and the amplification products (several copies per molecule) in the supernatant of the reaction can be collected. Variation of this approach can be implemented in any of the embodiments herein, including embodiments involving reverse transcription of a 3’ or 5’ tethered RNA and embodiments involving RNA-templated ligation of probes on a 3’ or 5’ tethered RNA. [0334] An overview for spatial transcriptomic profiling of FFPE tissue sections mediated by tethering of the 3’ end of a target mRNA molecule to a hydrogel via a hydrogel cleavable linker in accordance with some embodiments is depicted in FIG. 12A. In some^ny-2785575 202412020440 embodiments, an FFPE tissue section may be provided on a slide. In some embodiments, the tissue section may be baked, dewaxed and/or rehydrated. In some embodiments, an H&E stain may be applied and/or the tissue sample may be imaged, optionally using bright field microscopy (e.g., BF imaging). In some embodiments, the biological sample may be destained. The hydrogel (e.g., hydrogel mesh or hydrogel matrix) formation step may be carried out to intermesh (e.g., embed) the biological sample. During the formation of the sample intermeshed hydrogel (e.g., sample hydrogel), RNA molecules may be 3’ tethered to the hydrogel matrix by a cleavable linker. During the formation of the sample intermeshed hydrogel (e.g., sample hydrogel), RNA molecules may be 5’ tethered to the hydrogel matrix by a cleavable linker. In some embodiments, the cleavable linker comprises a cleavable segment. In some embodiments, the cleavable segment may comprise a disulfide bond. The tissue may be decrosslinked after tethering of the target molecule (e.g., target RNA or target nucleic acid) to the hydrogel matrix. In some embodiments, the tissue sample or components of the tissue sample may be removed by a clearing process leaving the RNA tethered (e.g., target molecule) to the hydrogel matrix. An RNA templated ligation probe (e.g., RTL probe) hybridization may be performed on the RNA tethered to the hydrogel. In the first step of RTL probe hybridization, in accordance with some embodiments, two probes including a left-hand side (LHS) probe and a right-hand side (RHS) probe may be hybridized to an mRNA target site of the tethered RNA as depicted in FIG. 12B. The LHS probe may include, in part, a Read 2S segment starting at its 5’ end. The RHS probe may include, in part, a poly(A) segment ending at its 3’ end as depicted in FIG. 12B. In some embodiments, following the probe hybridization step may be a probe ligation step. In some embodiments, the slide holding the ligated RNA product tethered to the hydrogel may be sandwiched to a hydrogel barcoded capture array. In some embodiments, capture probes (e.g., capture agents) of the hydrogel barcoded capture array may be immobilized to a second hydrogel intermeshed (e.g., second hydrogel mesh) with the capture probe of the barcoded capture array. In some embodiments, each capture probe may comprise a linker and an oligonucleotide sequence comprising a Read 1 sequence, a spatial barcode sequence, a UMI sequence, a poly(dT)VN sequence, or any combination of, as depicted in FIG. 12C. In some examples, the capture probe may capture the RNA target by hybridization of the poly(dT)VN sequence to the poly(A) section of the ligation product as depicted in FIGS. 12A and 12C. Following capture, in^ny-2785575 202412020440 accordance with some embodiments, the probe elongation and/or library preparation steps may be performed. [0335] In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA from the biological sample is tethered to a matrix of the hydrogel; b) digesting and removing the embedded biological sample from the hydrogel; c) releasing the target RNA from the matrix of the hydrogel; d) hybridizing the released target RNA to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; e) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the target RNA is tethered to the hydrogel matrix via a functional group at a 3’ end of the target RNA. In some embodiments, the target RNA is tethered to the hydrogel matrix via a functional group at a 5’ end of the target RNA. [0336] Turning to FIG. 13A, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh- frozen tissue sample) with the steps of 1) H&E staining the tissue sample; 2) imaging the tissue sample (e.g., using brightfield (BF)); and/or 3) destaining the tissue sample (1301). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1302). In some embodiments, the method further comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1303). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1304). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and a ligated probe is bound to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1305). In some embodiments, the method further comprises releasing the target RNA (e.g., by cleaving cleavable linkers or by changing the pH of a boronic acid-based hydrogel) for capture on the array (e.g., hydrogel barcoded capture array) (1306). In some embodiments, the method further comprises 1) reverse transcribing the target RNA captured on the array (e.g.,^ny-2785575 202412020440 hydrogel barcoded capture array) and 2) performing template switching and second-strand synthesis (1307) to generate a spatially labeled polynucleotide disclosed herein. In some embodiments, the method further comprises 1) performing amplification of a spatially labeled polynucleotide, e.g., amplification of the second strand synthesis product, and 2) proceeding with library preparation (1308). [0337] In some embodiments, provided herein is a method that comprises embedding the biological sample in a hydrogel matrix, wherein a target RNA may be tethered to the hydrogel matrix; removing the embedded biological sample from the hydrogel matrix; releasing the target RNA from the hydrogel matrix; d) hybridizing or ligating the released target RNA to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the target RNA may be 3’ tethered to the hydrogel matrix. In some embodiments, the target RNA may be 5’ tethered to the hydrogel matrix. In some embodiments, the capture domain in the capture probe comprises a 3’ poly(dT) sequence as depicted in FIG. 13B, and the poly(A) tail of the released target RNA hybridizes to the 3’ poly(dT) sequence. In some embodiments, the method comprises extending the capture probe by a polymerase (e.g., a reverse transcriptase) using the captured target RNA as a template, thereby generating an extended capture probe comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released target RNA as depicted in FIG. 13B. In some embodiments, the extended capture probe as depicted in FIG. 13B may further comprise a 3’ end homopolymer sequence, such as a poly(C) sequence. In some embodiments, the method may further comprise hybridizing a template switch oligonucleotide to the extended capture probe (e.g., via hybridization of rGrGrG to the CCC sequence in the extended capture probe, as shown in FIG. 13C), and extending the extended capture probe using the template switch oligonucleotide as a template to generate a further extended capture probe. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate.^ny-2785575 202412020440 [0338] As depicted in FIG. 13C, the method may further comprise hybridizing a primer (“Second Strand Primer” as shown in the figure) to the further extended capture probe at a sequence complementary to the template switch oligonucleotide (TSO), and extending the primer using the further extended capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the released target RNA and a complementary sequence of the spatial barcode as depicted in FIG. 13C. In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the further extended capture probe for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the array may be analyzed by nucleic acid sequencing. In some embodiments, after second strand-synthesis an Exo I treatment can be performed to get rid of the unused capture probes, followed by amplification. Exo I treatment prevents undesired priming of extended spatially labeled polynucleotides with proximal capture probes available on the surface of the array during amplification. [0339] In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA is tethered to a matrix of the hydrogel matrix; b) removing the embedded biological sample from the hydrogel; c) hybridizing a probe that is substantially complementary to a portion of the target RNA tethered to the matrix of the hydrogel; d) extending the hybridized probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) releasing the extended probe from the hydrogel; f) capturing the extended probe to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; and g) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. [0340] Turning to FIG. 14A, a method using a TSO-based capture approach for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g., a fresh-frozen tissue sample) with the steps of 1) H&E staining the tissue sample; 2) imaging the tissue sample (e.g., using brightfield (BF)); and/or 3) destaining the tissue sample (1401). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1402). In some embodiments, the method further^ny-2785575 202412020440 comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1403). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1404). In some embodiments, the method further comprises reverse transcribing the tethered RNA within matrix (e.g. while tethered to hydrogel) (1405). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel where a target RNA is tethered to the hydrogel and cDNA derived from the tethered RNA is bound to the tethered RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1406). In some embodiments, the method further comprises 1) releasing the cDNA and/or the target RNA from the matrix by a) reversing linker or dissolving matrix, or b) digesting target RNA tethered to matrix (e.g. using RNase H), and 2) capturing released cDNA and/or target RNA on an array such as a hydrogel barcoded capture array (e.g., using poly(rG) as a capture sequence) (1407). In some embodiments, the method further comprises performing on the captured cDNA and/or target RNA template switching and transcript extension using template switch oligo (TSO) priming (1408). In some embodiments, the method further comprises 1) performing amplification of the spatially labeled polynucleotide, e.g., a product of the captured target RNA and/or a product of the cDNA, and/or 2) proceeding with library preparation (1409). [0341] In some embodiments, provided herein is a method that comprises: embedding the biological sample in a hydrogel matrix, wherein a target RNA may be tethered to the hydrogel matrix; removing the embedded biological sample from the hydrogel matrix; hybridizing a probe that may be substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; extending the hybridized probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe and releasing the extended probe from the hydrogel matrix. In some embodiments, the method further comprises hybridizing or ligating the extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof as depicted in FIG. 14. In some embodiments, the probe hybridizes to a poly(A) sequence in the^ny-2785575 202412020440 target RNA tethered to the hydrogel matrix. In some embodiments, the probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence as depicted in FIG. 14. In some embodiments, the capture domain in the capture probe comprises a template switch oligonucleotide sequence and a homopolymer sequence (e.g., rGrGrG) complementary to the homopolymer sequence (e.g., CCC) at the 3’ end of the extended probe as depicted in FIG. 14. In some embodiments, the method may further comprise extending the extended probe using the capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a complementary sequence of the released target RNA and a complementary sequence of the spatial barcode as depicted in FIG. 14. In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the capture probe for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the spatial array may be analyzed by nucleic acid sequencing. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. [0342] In some examples, provided herein are methods for the spatial transcriptomic profiling of fresh frozen (e.g., FF) tissue sections mediated by 5’ target tethering to a hydrogel, utilizing a 5’ RT-based gene expression analysis and TSO-based capture. In some embodiments, an FF tissue section may be provided on a slide. In some embodiments, the tissue section may be H&E stained, imaged with a bright field configured microscope and/or destained. In some embodiments, a hydrogel (e.g., hydrogel matrix) may be formed within the tissue section. RNA target molecules (e.g., target RNA, target nucleic acid) may be tethered to the hydrogel matrix by a cleavable linker. In some examples, the cleavable linker may comprise a cleavable segment. In some examples, the cleavable segment may comprise a disulfide bond. In some examples, a clearing step (e.g., tissue removal) may be performed leaving target molecules tethered to the hydrogel. In some examples, following clearing, a reverse transcription step may be performed on the biological sample intermeshed with the sample gel to reverse transcribe the tethered RNA target molecule. In some embodiments, a sequence containing poly(dT) and non-poly(dT) sequences may be extended by reverse transcription. The reverse transcription product is then captured by the capture probe immobilized to the spatial array to generate a spatially labeled polynucleotide. Sequencing of the spatially labeled polynucleotide or a complement thereof^ny-2785575 202412020440 allows for association of the spatial barcode to the target molecule at a location in the biological sample, thereby revealing a spatial profile of the target molecule in the biological sample. [0343] In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA from the biological sample is tethered to a matrix of the hydrogel; b) removing the embedded biological sample from the hydrogel; c) hybridizing a first probe that is substantially complementary to a portion of the target RNA tethered to the matrix of the hydrogel; d) extending the hybridized first probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) hybridizing a second probe to the extended probe in the hydrogel and further extending the extended probe using the second probe as a template, wherein the further extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe; c) releasing the further extended probe from the hydrogel; d) capturing the further extended probe by a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; e) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the further extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. [0344] Turning to FIG. 15, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh- frozen tissue sample) with the steps of 1) H&E staining the tissue sample; 2) imaging the tissue sample (e.g., using brightfield (BF)); and/or 3) destaining the tissue sample (1501). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1502). In some embodiments, the method further comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through 3’ tethering of target nucleic acid via an optional linker) (1503). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1504). In some embodiments, the method further comprises reverse transcribing the tethered RNA within matrix (e.g. while tethered to hydrogel) (1505). In some embodiments, the method further comprises transcript switching oligo (TSO) priming. In some embodiments, the method further comprises contacting a transcript switching oligo (TSO) primer to the resulting cDNA within the matrix^ny-2785575 202412020440 (1506). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and cDNA is bound to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1507). In some embodiments, the method further comprises releasing the cDNA and/or target RNA by digesting the target RNA tethered to matrix (e.g. using RNase H) (1508). In some embodiments, the method further comprises performing second strand synthesis on the cDNA hybridized to the array (e.g. the matrix barcoded capture array) (1509). In some embodiments, the method further comprises performing amplification of a spatially labeled polynucleotide, e.g., amplification of the product of second strand synthesis of the captured cDNA, and 2) proceeding with library preparation (1510). [0345] In some embodiments, provided herein is a method that comprises: embedding the biological sample in a hydrogel matrix, wherein a target RNA from the biological sample may be tethered to the hydrogel matrix; removing the embedded biological sample from the hydrogel matrix; hybridizing a first probe that may be substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; extending the hybridized probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe, and hybridizing a second probe to the extended probe in the hydrogel matrix and further extending the extended probe using the second probe as a template, wherein the extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe. In some embodiments, the method may further comprise releasing the further extended probe from the hydrogel matrix. In some embodiments, the released further extended probe comprises the double stranded region or a portion thereof. In some embodiments, the released further extended probe is single stranded, e.g., when RNase H is used to digest the RNA strand (which is tethered to the hydrogel matrix) in the duplex which comprises a RNA:DNA hybrid, the DNA strand can be released from the hydrogel matrix as a single stranded further extended probe. In some embodiments, the method may further comprise hybridizing or ligating the further extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode. In some embodiments, the method may further comprise determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the further^ny-2785575 202412020440 extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, as depicted in FIG. 15. In some embodiments, the first probe hybridizes to a poly(A) sequence in the target RNA tethered to the hydrogel matrix as depicted in FIG. 15. In some embodiments, the first probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence. In some embodiments, the second probe comprises a 5’ poly(U) sequence, a template switch oligonucleotide sequence, and a homopolymer sequence (e.g., rGrGrG) complementary to the homopolymer sequence (e.g., CCC) at the 3’ end of the extended probe. In some embodiments, the homopolymer sequence comprises poly(rG). In some embodiments, the further extended probe comprises a 3’ poly(A) sequence, which is generated using the 5’ poly(U) sequence as a template. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. [0346] In some embodiments, the releasing comprises releasing the tethered RNA from the hydrogel matrix. In some embodiments, the releasing comprises releasing the further extended probe from the target RNA. In some embodiments, the releasing comprises contacting the hydrogel matrix with an RNase H as depicted in FIG. 15. In some embodiments, the capture domain in the capture probe comprises a 3’ poly(dT) sequence. In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising: i) from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released further extended probe, or ii) from 5’ to 3’: a sequence of the released further extended probe and a complementary sequence of the spatial barcode. In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the array for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the array may be analyzed by nucleic acid sequencing. D. Spatial Profiling of Open Chromatin, Protein Binding to Chromatin, and Chromatin Modifications [0347] A method disclosed herein can be used not only in gene expression profiling (e.g., as described in Section II-C), but also in spatial chromatin profiling, such as spatial ATAC which combines the assay for transposase-accessible chromatin and sequencing (ATAC-seq). The gene expression profiling and spatial ATAC-seq can be performed separately (e.g., on 109^ny-2785575 202412020440 separate biological samples or in separate regions of interest in the same biological sample), or the gene expression profiling and spatial ATAC-seq can be combined in one assay run performed on the same biological sample or the same region of interest thereof. [0348] In some embodiments, disclosed herein is a method for profiling open chromatin in a biological sample, comprising: a) contacting the biological sample with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence that is complementary to a capture domain of a capture probe on a spatial array, (ii) a linker sequence, and (iii) a reactive group; b) tagmenting the open chromatin with the plurality of transposomes; c) embedding the biological sample in a hydrogel and tethering the tagmented open chromatin to the hydrogel via the reactive group of the tagmented open chromatin; d) removing the biological sample from the hydrogel; e) releasing the tagmented open chromatin from the hydrogel and hybridizing the released tagmented open chromatin to the capture domain of the capture probe on the spatial array, wherein the capture probe further comprises a spatial barcode; and f) determining the profile of the open chromatin in the biological sample by determining (i) the sequence of all or a portion of the tagmented open chromatin, or a complement thereof, and (ii) the sequence of all or a portion of the spatial barcode, or a complement thereof. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. [0349] Turning to FIGS. 16A-16F, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh-frozen tissue sample) with the steps of 1) fixation of the tissue sample; 2) H&E or immunofluorescence (IF) staining of the tissue sample; 3) imaging the tissue sample (e.g., using brightfield (BF) or fluorescence imaging); 4) destaining the tissue sample; and/or 5) permeabilization of the tissue sample (e.g. gentle permeabilization) (1601). In some embodiments, the method further comprises preparing and/or activating RNA targets within the tissue sample for 3’ or 5’ tethering (e.g. “polishing” ends of RNA for attachment to boronic acid moieties) (1602). In some embodiments, the method further comprises performing transposition on targets (e.g., genomic DNA) within tissue sample (e.g., ‘tagmentation’ using Tn5) (1603). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a^ny-2785575 202412020440 hydrogel) (1604). In some embodiments, the method further comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1605). In some embodiments, the method further comprises decrosslinking the tissue sample from the matrix (1606). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1607). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared sample (e.g., a “tissue” hydrogel where a target RNA is tethered to the hydrogel) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1608). In some embodiments, the method further comprises releasing the target RNA tethered to matrix (e.g. using RNase H) and capturing on array (e.g., hydrogel barcoded capture array) (1609). [0350] In some embodiments, provided herein is a method wherein a biological sample is contacted with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence that may be complementary to a capture domain of a capture probe on the array, (ii) a linker, and (iii) a reactive group, thereby tagmenting an open chromatin in the biological sample with the plurality of transposomes, and wherein the tagmented open chromatin may be tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin, wherein after the biological sample may be removed from the hydrogel matrix, the tagmented open chromatin may be released from the hydrogel matrix, the released tagmented open chromatin may be hybridized or ligated to a capture domain of a capture probe on the array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof may be determined. [0351] An overview of the method for analyzing a biological sample, in accordance with some embodiments, may comprise spatial ATAC and 3’ or 5’-gene expression workflow as depicted in FIG. 16A. In some embodiments, the workflow may comprise the steps of: fixing the FF tissue section; H&E/IF staining; imaging, destaining; permeabilizing the biological sample; preparing and activating the RNA target for 3’ or 5’ tethering; tagmentation (e.g., using a Tn5 transposome); hydrogel matrix embedding (during and/or after which the target RNA molecules and transposed DNA can be tethered to the hydrogel matrix); descrosslinking the tissue in the hydrogel matrix; removing the tissue (e.g., by clearing), while the target molecules (e.g., target 111^ny-2785575 202412020440 RNA molecules and transposed DNA) remain tethered to the hydrogel matrix; and simultaneously releasing the target molecules (e.g., target RNA molecules and transposed DNA) from the sample hydrogel matrix and capturing the released molecules with the hydrogel barcoded capture array. In some embodiments, target RNA molecules for the RNA-seq modality and transposed DNA for the ATAC-seq modality are captured by different capture probes on the hydrogel barcoded capture array, for instance, as shown in FIG. 19. [0352] In some embodiments, the permeabilization step may be carried out gently to facilitate the RNA target preparation step. In some embodiments, after the clearing step the target molecules (e.g., target RNA molecules and transposed DNA) remain in the hydrogel. The target releasing step may comprise contacting the sample hydrogel (e.g., tissue hydrogel, substrate or first substrate) with the hydrogel barcoded capture array (e.g., second substrate). The contacting step may also be referred to as the sandwiching step. Tethered target molecules (e.g., target RNA molecules and transposed DNA) may be released by cleaving a cleavable linker. The cleavable linker may comprise a disulfide bond that may be chemically cleaved. In some embodiments, following release and capture of target molecules (e.g., target RNA molecules and transposed DNA), workflow may proceed with a step of amplifying material and/or generating libraries. Subsequent analysis may then be performed, which may in some instances include associating the spatial barcode with locations in the biological sample, thereby identifying the presence of the target molecules (e.g., target RNA molecules and transposed DNA) at locations in the biological sample. [0353] FIGS. 16B-16D depict an exemplary transposition step where, in certain circumstances a transposome complex, in this example a transposome comprising a Tn5 transposase and modified transposon ends, may be modified for tethering of transposed DNA. A modified design for a transposon end in the transposome complex, in accordance with some embodiments, is depicted in FIG. 16B. In certain aspects, the transposon end (e.g., mosaic end) may comprise a PCR handle (pR2), a cleavable (e.g., disulfide or photo-cleavable) linker, and a 5’-acrydite at its terminus. The acrydite functionality may be a reactive group for hydrogel embedding. In some embodiments, the disulfide linker may allow for chemically triggered release (e.g., by treatment of a reducing agent such as dithiothreitol (DTT)) of the tethered target molecule (e.g., the transposed DNA). Assembly of a partial capture sequence and Tn5 mosaic end duplex with the PCR handle (pR2) and Tn5 transposase, in accordance with some^ny-2785575 202412020440 embodiments, is depicted in FIG. 16C. The structure of the transposed DNA, in accordance with some embodiments, is depicted in FIG. 16D. In some embodiments, the acrydite group allows for tethering of a transposed DNA (e.g., from an open chromatin region) to the sample hydrogel. Any transpositional system can be equally applicable, for example a Mu, Vibrio, Marinar, Tn7, or other transpositional system once modified could also be used to tagment the accessible chromatin and used in tethering methods as described herein. [0354] The structures of capture probes on the hydrogel barcoded array, in accordance with some embodiments, are depicted in FIGS. 16E-16F. For example, one of the capture domains may be specific to capturing RNA or an RNA proxy using a poly(T) capture domain. The second capture domain may be specific to a mosaic end of a transposon, such that tagmented accessible DNA is captured concurrently by those specific capture domains of capture probes on an array. As such, more than one target nucleic acid can be captured concurrently on the sample array depending on the capture domain sequences on the array, thereby determining the spatial expression patterns of, in this example, RNA or a proxy thereof as well as accessible chromatin from a sample. FIG. 7 provides examples of different capture domains that could be used for different target nucleic acid capture from a biological sample, such as using a homopolymer capture domain, a fixed sequence capture domain, or a degenerate or partially degenerate capture domain. [0355] In some embodiments, a molecule comprising transposed DNA of the ATAC- seq modality can be captured (via the partial capture sequence) by the capture probe comprising the spacer shown in FIG. 16E. In one example, the spacer in the capture probe can comprise a sequence substantially complementary to a sequence in the partial capture sequence of the transposed DNA, such that the spacer can hybridize to the partial capture sequence, followed by ligation with or without gap filling prior to the ligation. In one example, a splint oligonucleotide comprising a first sequence substantially complementary to the spacer in the capture probe and a second sequence substantially complementary to the partial capture sequence in the transposed DNA can be used to connect the capture probe to the transposed DNA by ligation using the splint oligonucleotide as a template, with or without gap filling prior to the ligation. A molecule comprising a transcript sequence or complement thereof of the RNA-seq modality can be captured (via the poly(A) sequence) by the capture probe comprising a poly(dT) sequence shown in FIG. 16F.^ny-2785575 202412020440 [0356] Turning to FIGS. 17A-17D, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh-frozen tissue sample) with the steps of 1) fixation of the tissue sample; 2) H&E or immunofluorescence (IF) staining of the tissue sample; 3) imaging the tissue sample (e.g., using brightfield (BF) or fluorescence imaging); 4) destaining the tissue sample; and/or 5) permeabilization of the tissue sample (e.g. gentle permeabilization) (1701). In some embodiments, the method further comprises preparing and/or activating RNA targets within the tissue sample for 3’ or 5’ tethering (e.g. “polishing” ends of RNA for attachment to boronic acid moieties) (1702). In some embodiments, the method further comprises performing primary and secondary antibody incubations on the tissue sample (1703). In some embodiments, the method further comprises performing transposition on tissue sample (e.g. using pA-Tn5) (1704). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1705). In some embodiments, the method further comprises decrosslinking the tissue sample from the matrix (1706). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1707). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared sample (e.g., a “tissue” hydrogel where a target RNA is tethered to the hydrogel) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1708). In some embodiments, the method further comprises releasing the target RNA tethered to matrix (e.g. by changing the pH of a boronic acid-based hydrogel) and capturing the target RNA on the array (e.g., hydrogel barcoded capture array) (1709). [0357] In some embodiments, provided herein is a method wherein a biological sample, in accordance with some embodiments, is contacted with an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, and with a transposome-binding moiety complex that may bind to the antibody, wherein the transposome- binding moiety complex may comprise (i) a transposase (e.g., Tn5), (ii) an antibody-binding moiety (e.g., protein A), (iii) a transposon end sequence comprising a capture sequence that may be complementary to a capture domain of a capture probe on a spatial array, (iv) a reactive group, or a combination thereof, thereby tagmenting a genomic DNA in the biological sample with the aide of the transposome-binding moiety complex. In some embodiments, the^ny-2785575 202412020440 transposome-binding moiety complex comprises a transposone and a binding moiety. In such embodiments, the tagmented genomic DNA may be tethered to the hydrogel matrix via the reactive group of the transposome-binding moiety complex, wherein after the biological sample may be removed from the hydrogel matrix, the tagmented genomic DNA may be released from the hydrogel matrix, the released tagmented genomic DNA may be hybridized or ligated to a capture domain of a capture probe on the spatial array. In some embodiments, the capture probe may further comprise a spatial barcode. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. In such embodiments, the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof may be determined. [0358] Exemplary transposome-binding moiety complexes include but are not limited to the transposome-antibody-binding moiety complexes described in US 2023/0175045 A1 and US 2023/0287481 A1, which are incorporated herein by reference in their entireties. [0359] In some embodiments, ChIP-seq may provide information on genome-wide DNA binding sites, associated DNA-binding proteins (e.g., transcription factors/co-factors), localization of histones and/or their modifications throughout the genome or any combination thereof. In some embodiments, combined with gene expression profiling, ChIP-seq may allow for investigating the mechanism of gene regulation. [0360] In some examples, simultaneous release and/or capture of genomic domains may be bound by a specific protein along with transcripts in a single step (e.g., same experimental run). In some embodiments, target molecules may be immobilized to a hydrogel matrix (e.g., sample hydrogel mesh). In certain circumstances, such a strategy may allow for the release of targets separately from (e.g., after) the tissue permeabilization step. In some embodiments, retention of the target molecules during the permeabilization step and release of the target molecules after permeabilization may occur at a time chosen by the user. [0361] In some examples, transcript tethering may comprise a modified oligonucleotide of the pA-transposome assembly, the modified oligonucleotide comprising: a reactive group (e.g., an acrydite) for tethering of the transposed DNA sequence in proximity of^ny-2785575 202412020440 the target, a cleavable linker for controlled release of the target molecule over time, or any combination thereof. [0362] In some embodiments, after target tethering, the tissue may be fully digested to remove unwanted tissue related structures and molecules. In some embodiments, the tissue sample may be removed after digesting. In some embodiments, the tethered target molecules (e.g., target molecules tethered to the hydrogel matrix, where the hydrogel matrix is on the slide from which the tissue sample has been removed), may be sandwiched between the barcoded solid support (e.g., an array with or without a hydrogel on it) and the slide on which the tissue sample is place as depicted in FIG. 17A. In the hydrogel-based sandwich assembly in accordance with some embodiments, target molecules can be controllably released from the sample hydrogel and captured simultaneously, thereby associating the target molecule’s presence, absence, or quantity to a location in the sample. [0363] An exemplary workflow is provided in FIG. 17A. The workflow applied to an fresh frozen tissue section may comprise the following steps: fixing; H&E/IF staining; imaging; destaining; gently permeabilizing the biological sample to facilitate the next step of preparing the RNA target for 3’ or 5’ tethering; 3’ or 5’ tethering; incubating the primary and secondary antibodies; transposing accessible genomic DNA via the pA-Tn5 transposome complex; hydrogel matrix embedding (during and/or after which the target RNA molecules and transposed DNA can be tethered to the hydrogel matrix); descrosslinking the tissue in the hydrogel matrix; removing the tissue (e.g., by clearing), while the target molecules (e.g., target RNA molecules and transposed DNA) remain tethered to the hydrogel matrix; and simultaneously releasing the target molecules (e.g., target RNA molecules and transposed DNA) from the sample hydrogel matrix and capturing the released molecules with the hydrogel barcoded capture array. In some examples, sandwiching the sample hydrogel and hydrogel barcoded capture array may be carried out. In some examples, the tethered target molecules (e.g., target RNA molecules and transposed DNA) may be released by cleaving a disulfide linker. In some examples, following target release and capture, the workflow may proceed to amplify material and/or generate libraries. In some embodiments, target RNA molecules for the RNA-seq modality and transposed DNA for the ChIP-seq modality are captured by different capture probes on the hydrogel barcoded capture array, for instance, as shown in FIG. 19.^ny-2785575 202412020440 [0364] FIGS. 17B-17D provide an exemplary workflow of the transposing step in accordance with some embodiments. In some embodiments, the method may comprise antibody- based binding of the target DNA-binding protein or histone modification of interest; secondary antibody binding which can enhance the tethering of protein A (pA)-Tn5 transposome; fragmenting and tagging accessible chromatin close to the protein binding site and appending adapter DNA sequences; or any combination thereof. The target DNA-binding protein, primary antibody, secondary antibody and pA-Tn5 transposome comprising modified transposon end sequences, in accordance with some embodiments are depicted in FIG. 17B. A partial capture sequence and Tn5 mosaic end duplex, pA-Tn5 transposase and a PCR handle (pR2) in accordance with some embodiments are depicted in FIG. 17C. In some embodiments, a cleavable linker can connect an acrydite functionality at the 5’ end to the PCR handle (pR2). The cleavable linker in some examples may comprise a disulfide bond. The acrydite functionality may be utilized for tethering the transposed DNA to the sample hydrogel. The structure of the transposed DNA in accordance with some embodiments is depicted in FIG. 17D. E. Spatial Profiling of Proteins [0365] In some embodiments, disclosed herein is a method for spatial profiling of a non-nucleic acid analytes, such as proteins, for example, by using an analyte capture agent that binds to the non-nucleic acid analyte and comprises an analyte associated oligonucleotide (e.g., comprising an analyte binding moiety barcode such as an antibody barcode) that corresponds to or is associated with the identity or a feature of the non-nucleic acid analyte (e.g., an antigen/epitope binding specificity of an antibody). [0366] In some embodiments, the biological sample is contacted with an analyte capture agent comprising (i) an analyte-binding moiety that binds to a non-nucleic acid analyte in a biological sample, (ii) an analyte capture sequence configured to be captured by a capture domain of a capture probe on a spatial array, (iii) an analyte binding moiety barcode corresponding to or associated with the analyte-binding moiety and/or the analyte or a portion thereof, and (iv) a reactive group. In some embodiments, the biological sample is embedded in a hydrogel matrix, wherein the analyte capture agent or a portion thereof is tethered to the hydrogel matrix via the reactive group of the analyte capture agent. In some embodiments, at least the analyte associated oligonucleotide (comprising the analyte capture sequence and the^ny-2785575 202412020440 analyte binding moiety barcode) or a portion thereof of the analyte capture agent is tethered to the hydrogel matrix. [0367] In some embodiments, after sample removal from the hydrogel matrix (e.g., via sample clearing), the tethered analyte capture agent or portion thereof is released from the hydrogel matrix, and the released analyte capture agent or portion thereof is hybridized or ligated to the capture domain of the capture probe. In some embodiments, the analyte capture sequence of the analyte capture agent hybridizes to the capture domain of the capture probe, thereby capturing the analyte capture agent to the capture probe on the spatial array. In some embodiments, the analyte capture sequence of the analyte capture agent hybridizes to a splint oligonucleotide which in turn hybridizes the capture domain of the capture probe. The capture sequence and the capture domain can be covalently linked via ligation, with or without gap- filling prior to ligation using the splint oligonucleotide as a template. Using nucleic acid hybridization and/or ligation, the analyte capture sequence and the capture probe can be linked to allow integration of sequence information of the analyte binding moiety barcode (of the analyte capture agent) and the spatial barcode (of the capture probe) in the same nucleic acid molecule (which is a spatially labeled polynucleotide). [0368] In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. [0369] In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising (i) a sequence of the analyte binding moiety barcode in the analyte capture agent, or a complementary sequence thereof, and (ii) a sequence of the spatial barcode, or a complementary sequence thereof. The spatially labeled polynucleotide or a portion thereof can be released from the array for analysis, for instance, by nucleic acid sequencing, such as sequencing by synthesis, sequencing by ligation, sequencing by binding, sequencing by avidity, sequencing by hybridization, or any combination thereof. [0370] In some embodiments, a reactive group for hydrogel embedding and a cleavable linker for subsequent cleavage and release of the analyte capture agent or a portion thereof from the hydrogel matrix can be appended to the analyte capture agent. The cleavable linker can comprise a restriction site, a disulfide bond, a photo-cleavable bond or group, or any combination thereof.^ny-2785575 202412020440 [0371] In some embodiments, the analyte capture agent is an antibody- oligonucleotide conjugate comprising (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group. In some embodiments, an antibody can be covalent or noncovalently linked to an oligonucleotide comprising an analyte capture sequence, an analyte binding moiety barcode (e.g., an antibody barcode), an optional functional sequence (e.g., a primer binding sequence for PCR), and a cleavable linker. In some embodiments, the analyte capture agent is an antibody-oligonucleotide conjugate, wherein the analyte-associated oligonucleotide is the oligonucleotide of the antibody-oligonucleotide conjugate. In some embodiments, the barcode sequence associated with the antibody of the analyte capture agent is an analyte binding moiety barcode. The linker can be partially double stranded and partially single stranded, and can comprise one or more oligonucleotides, at leach one of which is coupled to a reactive group, such as 5’-acrydite or 3’-acrydite or an internal acrylate in the at least one oligonucleotide, for tethering to a hydrogel matrix. [0372] In some embodiments, an antibody can be covalent or noncovalently linked to an oligonucleotide, which hybridizes to another oligonucloetide comprising an analyte capture sequence, an analyte binding moiety barcode (e.g., an antibody barcode), and an optional functional sequence (e.g., a primer binding sequence for PCR). The oligonucleotide can comprise or be hybridized to a linker oligonucleotide coupled to a reactive group, such as 5’- acrydite, for tethering to a hydrogel matrix. [0373] Turning to FIGS. 18A-18F, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. an FFPE sample) with the steps of 1) baking the slide comprising the sample; 2) dewaxing the sample; 3) rehydrating the sample; 4) performing H&E staining; 5) imaging the sample (e.g. using brightfield (BF) imaging); and/or 6) destaining the sample (1801). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1802). In some embodiments, the method further comprises tethering a target (e.g., a target nucleic acid such as RNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1803). In some embodiments, the method further comprises decrosslinking the sample in the matrix (1804). In some embodiments, the method further^ny-2785575 202412020440 comprises contacting the tissue sample with a probe or probe set (e.g. a linear probe set) that directly or indirectly binds to the target tethered to the matrix, followed by one or more optional wash(es) (1805). In some embodiments, the method further comprises ligating the probe or probe set bound to the target within the matrix (e.g., RNA-templated ligation), followed by one or more optional wash(es) (1806). In some embodiments, the method further comprises 1) blocking the tissue sample within the matrix, and 2) incubating the tissue sample with a labelling agent (e.g., an oligonucleotide-tagged antibody) (1807). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while tethered target remains attached to matrix (1808). In some embodiments, the method further comprises sandwiching the matrix- embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and a ligated probe is hybridized to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1809). In some embodiments, the method further comprises releasing the target and/or the ligated probe (e.g., with RNase H treatment) for capture on the array (1810). In some embodiments, the method further comprises elongating the probe captured on array and proceeding with library preparation (1811). [0374] In some embodiments, provided herein is a method utilizing an analyte capture agent comprising (i) an analyte binding moiety (e.g., an antibody or epitope binding fragment thereof, aptamer, etc.) that binds to a target protein in the biological sample, (ii) an analyte capture sequence configured to be captured (e.g., via hybridization and/or ligation) by a capture domain of a capture probe on the array, (iii) an analyte binding moiety barcode associated with the antibody, (iv) a reactive group, or any combination thereof. In some embodiments, the analyte binding moiety is an antibody or antibody moiety. In some embodiments, the analyte binding moiety is an antibody or epitope binding fragment thereof. In some embodiments, the analyte binding moiety is an antibody that binds to a target protein in a biological sample. In some embodiments, a tagged aptamer targeting a protein of interest can be used, and the tag can comprising an analyte capture sequence and/or an analyte binding moiety barcode, and the tagged aptamer can comprise a reactive group. In some embodiments, after the biological sample is removed from the hydrogel matrix, the analyte capture agent or a portion thereof (e.g., at least a portion of the analyte capture sequence and at least a portion of the analyte binding moiety barcode) may be released from the hydrogel matrix and/or the analyte^ny-2785575 202412020440 capture sequence or a portion thereof (in the released analyte capture agent or portion thereof) may be hybridized or ligated to a capture domain of a capture probe on the array. In some embodiments, the capture probe may further comprise a spatial barcode, where the sequence of (i) the analyte binding moiety barcode associated with the antibody, or a complement of the analyte binding moiety barcode, and (ii) the spatial barcode, or a complement thereof, may be determined. The analyte capture agent can comprise a primary antibody that targets a protein or a portion thereof (e.g., an epitope of the protein, or a protein modification of interest), or a secondary antibody that binds the primary antibody. The analyte capture agent can further comprise an analyte binding moiety barcode which comprises a sequence that corresponds to, identifies, and/or is associated with the protein or portion thereof and/or an epitope binding region of the primary antibody. The analyte capture agent can further comprise a moiety that comprises or is directly or indirectly linked to a reactive group (e.g., an acrydite) for tethering to a hydrogel matrix. The antibody portion(s) of the analyte capture agent can be digested during tissue clearing/digestion and the analyte binding moiety barcode or a portion thereof is held in place (e.g., reversibly tethered to the hydrogel matrix via a boronate ester bond described in Section II (B) (ii), or tethered via a cleavable linker) until cleavage during target release (e.g., in the sandwich step). For multiplexing, a cocktail of analyte capture agents (e.g., antibody- oligonucleotide conjugates) can be used. [0375] A probe-based workflow for spatial protein and gene expression analysis of an FFPE biological sample in accordance with some embodiments is exemplified in FIG. 18A. In some embodiments, the workflow may comprise subjecting a FFPE tissue section to steps comprising: baking the tissue section; dewaxing; rehydrating; H&E staining; bright field imaging; destaining; forming the hydrogel including target tethering to the hydrogel; descrosslinking the tissue in the sample hydrogel; RTL probe hybridizing and washing; probe ligating and washing; tissue blocking and incubating with an antibody-oligo conjugate (e.g., analyte capture agent); tissue removing/clearing; sandwiching the sample hydrogel with the hydrogel barcoded capture array including the release of the RTL probe ligation product and/or the tethered target RNA and its capture on the array; probe elongation; library preparation; or any combination thereof. Subsequent analysis may then be performed, where the analysis may comprise associating the spatial barcode with a location in the biological sample, thereby identifying the presence of the target molecule at the location in the biological sample.^ny-2785575 202412020440 [0376] The analyte capture agent (e.g., an antibody-oligonucleotide conjugate), in accordance with some embodiments is depicted in FIG. 18B. The antibody-oligonucleotide conjugate may comprise a capture sequence configured to be hybridized or ligated to a capture probe on a hydrogel barcoded capture array. In some embodiments, the capture sequence in the antibody-oligonucleotide conjugate is substantially complementary to a capture region of the capture probe. The antibody-oligonucleotide conjugate may comprise one or more molecules of a conjugated antibody. In some embodiments, the antibody is conjugated to an analyte associated oligonucleotide comprising an analyte capture sequence (e.g., the “capture sequence” as shown in FIGS. 18B-18C), the analyte binding moiety barcode, and optionally a PCR handle (pR2). In some embodiments, the antibody is conjugated to a linker oligonucleotide that hybridizes to a linker sequence in the oligonucleotide (e.g., as shown in FIG. 18D). The antibody-oligonucleotide conjugate can comprise or be linked to a reactive group such as an acrydite group configured for tethering to the hydrogel. In some embodiments, the reactive group is linked via a linker (e.g., a cleavable linker) to an oligonucleotide which hybridizes to an intermediate oligonucleotide that in turn hybridizes to a linker sequence in the oligonucleotide conjugated to the antibody (e.g., as shown in FIG. 18B). In some embodiments, the reactive group is linked via a linker (e.g., a cleavable linker) to an oligonucleotide which hybridizes to a linker sequence in the oligonucleotide conjugated to the antibody (e.g., as shown in FIG. 18C). In some embodiments, the reactive group is linked via a linker (e.g., a cleavable linker) to an oligonucleotide which hybridizes to a first linker sequence in an analyte associated oligonucleotide comprising an analyte capture sequence (e.g., a spacer as shown in FIG. 18D) and an analyte binding moiety barcode (e.g., an antibody-specific barcode as shown in FIG. 18D), where the antibody is conjugated to a linker oligonucleotide that hybridizes to a second linker sequence in the analyte associated oligonucleotide (e.g., as shown in FIG. 18D). The first and second linker sequences in the analyte associated oligonucleotide can be adjacent to each other. In some embodiments, the first and second linker sequences are at the 3’ end of the analyte associated oligonucleotide. [0377] In some embodiments, to allow for reversible tethering, a cleavable linker may comprise a disulfide bond. In some embodiments, in an analyte capture agent, the antibody portion targeting a protein of interest at a location in the biological sample may be digested during the tissue clearing/digestion step. In some embodiments, the analyte associated^ny-2785575 202412020440 oligonucleotide portion of the analyte capture agent is held in place until the release step. In some examples, the release may be triggered by chemically cleaving the disulfide bond of the cleavable linker. [0378] In some embodiments, the analyte capture agent comprises an internal acrylate between the cleavable region and the 3’ end of the oligonucleotide as depicted in FIG. 18C. In some embodiments, the analyte capture agent comprises a cleavable linker, a hydrogel tethering moiety, and a spacer as depicted in FIG. 18D. In some embodiments, the capture probe may comprise a capture region configured to capture the analyte capture agent or a portion thereof. In some embodiments, the capture probe as depicted in FIG 18E is configured to capture the analyte capture agent or a portion thereof depicted in FIGS. 18B-18C. In some embodiments the capture probe depicted in FIG. 18E is configured to capture a analyte capture agent or a portion thereof using a hybridization and extension approach. In some embodiments, the capture probe depicted in FIG. 18F may be configured to capture the analyte capture agent or a portion thereof as depicted in FIG. 18D. In some embodiments, the capture probe as depicted in FIG. 18F may be configured to capture the analyte capture agent or a portion thereof using a ligation/gap filling approach. [0379] In some embodiments, an analyte capture agent may include an analyte binding moiety that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the analyte capture agent can comprise an analyte binding moiety barcode that is indicative of the analyte or portion thereof interacting with an analyte-binding moiety of the analyte capture agent. For example, an analyte binding moiety barcode may comprise a barcode sequence that permits identification of the analyte being tagged, such as a barcode that binds a protein. In some embodiments, the analyte capture agent comprises an analyte binding moiety and an analyte binding moiety barcode domain comprising one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety and/or the analyte. In some embodiments, an analyte binding moiety barcode includes a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds is identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and/or sequence that is associated with the analyte^ny-2785575 202412020440 binding moiety. An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein. [0380] In some embodiments, the method comprises one or more post-fixing (also referred to as post-fixation) steps after contacting the sample with one or more analyte capture agents. [0381] In the methods and systems described herein, one or more analyte capture agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes such as proteins, including proteins in cells or on the cell surface. Analytes may include, but are not limited to, a protein, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T- cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, analytes may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof. [0382] In some embodiments, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). An analyte binding moiety may include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro- body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The analyte capture agent can comprise an oligonucleotide that is indicative of the cell surface feature to which the analyte binding moiety binds. For example, the oligonucleotide may comprise a barcode sequence that permits identification of the analyte binding moiety. For example, an analyte binding moiety that is specific to af first protein may have coupled thereto a first oligonucleotide, while an analyte binding moiety that is specific to a different protein (e.g., a second protein) may have a different oligonucleotide coupled thereto. For a description of exemplary analyte capture agents, analyte binding moieties, associated oligonucleotides, and^ny-2785575 202412020440 methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969, all of which are herein incorporated by reference in their entireties. [0383] In some embodiments, an analyte binding moiety includes one or more antibodies or epitope-binding fragments thereof. The antibodies or epitope-binding fragments including the analyte binding moiety can specifically bind to a target analyte. In some embodiments, the analyte is a protein (e.g., a protein on a surface of the biological sample (e.g., a cell) or an intracellular protein). In some embodiments, a plurality of analyte capture agents comprising a plurality of analyte binding moieties bind a plurality of analytes present in a biological sample. In some embodiments, the plurality of analytes includes a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte capture agents are the same. In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte capture agents are the different (e.g., members of the plurality of analyte capture agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites). In some embodiments, the plurality of analytes includes multiple different species of analyte (e.g., multiple different species of polypeptides). [0384] In some aspects, an analyte associated oligonucleotide in an analyte capture agent comprises one or more nucleic acid barcode sequences that permit identification of an analyte binding moiety which the oligonucleotide is coupled to. The selection of oligonucleotides as the analyte associated oligonucleotides may provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using the spatial detection techniques described herein. [0385] Attachment (coupling) of the analyte associated oligonucleotides to the analyte binding moieties may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of an analyte binding moiety (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labeling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g.,^ny-2785575 202412020440 using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride- Cleavable Biotinylation Phosphoramidite for 5′-end-Labeling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which is entirely incorporated herein by reference for all purposes. Furthermore, click reaction chemistry may be used to couple oligonucleotides to analyte binding moieties. In another example, an analyte binding moiety is indirectly (e.g., via hybridization) coupled to an oligonucleotide comprising a barcode sequence that identifies the label agent. For instance, the analyte binding moiety may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that comprises a sequence that hybridizes with a sequence of the oligonucleotide. Hybridization of the hybridization oligonucleotide to the oligonucleotide couples the analyte binding moiety to the oligonucleotide. In some embodiments, the oligonucleotides are releasable from the analyte binding moiety, such as upon application of a stimulus. For example, the oligonucleotide may be attached to the analyte binding moiety through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. [0386] In some cases, the analyte capture agent can comprise an oligonucleotide and a label. A label can be a fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to an analyte capture agent (or associated oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the analyte capture agent or associated oligonucleotide). In some cases, a label is conjugated to a first oligonucleotide that is complementary (e.g., hybridizes) to a sequence of the analyte capture agent associated oligonucleotide. [0387] In some embodiments, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes can be associated with locations of the analytes in the biological sample. Such^ny-2785575 202412020440 information (e.g., proteomic information when the analyte binding moiety(ies) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (e.g., sequences of transcripts), or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte capture agent comprising an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety. Results of protein analysis in a sample (e.g., a tissue sample or a cell) can be associated with DNA and/or RNA analysis in the sample. F. Spatial Multiome Profiling of Analytes [0388] In some embodiments, any two or more of the methods disclosed herein can be combined, for instance, for spatial multiome profiling mediated by tethering analytes and/or proxies thereof to a matrix (e.g., a hydrogel matrix) embedding a biological sample to be analyzed, followed by sample removal from the matrix (e.g., by clearing at least a subset of proteins and/or lipids in the biological sample from the matrix). The analytes and/or proxies thereof that remain tethered to the matrix after sample removal can be released from the matrix, and captured on a spatial array for analysis. In some embodiments, the method comprises reversibly tethering target molecules into a hydrogel matrix and releasing the tethered target molecules for capture on an array. [0389] In some embodiments, a spatial multiome analyte profiling method disclosed herein can be used to detect one or more RNA species (e.g., transcriptomic profiling), one or more epigenetic modifications, one or more epitranscriptomic modifications, one or more DNA or RNA binding proteins, one or more single nucleotide variations (e.g., one or more SNPs or point mutations), one or more protein interactions and/or one or more post-translational modifications (e.g., using a proximity ligation/proximity extension assay), a high resolution imaging workflow, and/or expansion microscopy. [0390] In some embodiments, a spatial multiome analyte profiling method disclosed herein involves integration of different modalities (e.g., gene expression, protein profiling, ATAC-seq, and/ChIP-seq) by using a spatial array comprising capture probes comprising different capture domains, each different capture domain for analyzing one or more different^ny-2785575 202412020440 modalities. In some embodiments, a feature on a spatial array can comprise capture probes comprising different capture domains and the same spatial barcode. [0391] In some embodiments, provided herein are two or more or all of the released ligation product (e.g., a ligated linear probe as described in Section II(C) or Example 1), the released target RNA (e.g., as described in Section II-C or Example 2), the extended probe (e.g., as described in Section II-C, Example 3, or Example 4), and the further extended probe (e.g., as described in Section II-C, Example 3, or Example 4) are captured by capture domains of different sequences in the capture probes on the array. In some embodiments, two or more or all of the released ligation product, the released target RNA, the extended probe, and the further extended probe are each hybridized or ligated to a capture domain of a different sequence in the capture probes on the array, for instance, as shown in FIG. 19. In some embodiments, capturing comprises hybridizing, ligating, or a combination thereof. III. COMPOSITIONS AND KITS [0392] In some embodiments, disclosed herein is a composition or kit comprising one or more reagents for performing the methods provided herein, e.g., any described in Section II, for example reagents required for one or more steps comprising hybridization, ligation, primer extension, reverse transcription, amplification, detection, sequencing, and/or sample preparation as described herein. In some embodiments, any or all of the polynucleotides found in a composition or kit disclosed herein are DNA molecules. In some embodiments, the kit further comprises a kinase (e.g., a polynucleotide kinase like T4 DNA kinase). In some embodiments, the kit further comprises a ligase, for instance for ligating together two RTL probes. In some embodiments, the ligase has DNA-splinted DNA ligase activity. In some embodiments, the kit further comprises a polymerase, for instance for performing extension and/or amplification of the ligated probes or capture probe extensions for first and/or second strand synthesis from a captured target nucleic acid. In some embodiments, the kit further comprises a primer for amplification. [0393] In some embodiments, disclosed herein is a composition or kit comprising one or more reagents for array-based analysis, such as capture probes on a spatial array, e.g., any described in Sections I and II. For example, provided are kits comprising a substrate (e.g., an array) with a plurality of oligonucleotides immobilized thereon. For example, the oligonucleotides immobilized on the substrate each comprise a spatial barcode sequence and a^ny-2785575 202412020440 capture region configured to be hybridized or ligated to a sequence of a target RNA or a complement thereof, or a ligated probe or a complement thereof, or transposed DNA or a complement thereof, or an analyte capture agent (e.g., one that comprises an antibody specific to a protein, a capture sequence, and an antibody barcode associated with the antibody) or a complement thereof (e.g., as described in Section II-C through Section II-F). In some embodiments, the oligonucleotides immobilized on the substrate each comprises a spatial barcode sequence and a capture sequence complementary to a sequence of a ligated probe, an analyte capture agent capture sequence and/or a target nucleic acid such as mRNA. [0394] Disclosed herein in some aspects is a kit comprising a probe or a probe set, wherein the molecules of the probe or the probe set lack phosphorylated 5’ ends, the kit further comprising a kinase for phosphorylating one or more 5’ ends of the probe or the probe set hybridized to a target nucleic acid. [0395] In some embodiments, the kit further comprises reagents for extending the capture probes that have hybridized or ligated ligations products, target nucleic acids and/or analyte capture agent capture sequences from the biological sample, thereby generating extension products for further analysis. In some embodiments, the kit futher comprises reagents for generating sequencing libraries from the extension products for downstream sequencing. Such reagents can include, but are not limited to, one or more of a reverse transcriptase, a DNA polymerase, dNTPs, buffers, etc. [0396] The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the kits further contain instructions for using the components of the kit to practice the provided methods. [0397] In some embodiments, the kits can contain reagents and/or consumables required for performing one or more steps of the provided methods. In some embodiments, the kits contain reagents for fixing, embedding, and/or permeabilizing the biological sample. In some embodiments, the kits contain reagents, such as enzymes and buffers for ligation and/or amplification, such as ligases and/or polymerases. In some aspects, the kit can also comprise any one of the reagents described herein, e.g., wash buffer and ligation buffer. In some embodiments, the kits contain reagents for detection and/or sequencing. In some embodiments,^ny-2785575 202412020440 the kits optionally contain other components, for example nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, reagents for additional assays. [0398] In some embodiments, provided herein is a kit for analyzing a biological sample, the kit comprising: a matrix-forming agent for embedding the biological sample in a matrix; and an agent for tethering a target RNA of the biological sample to the matrix. In some embodiments, the matrix-forming agent is a hydrogel matrix-forming agent and forms a hydrogel embedding the biological sample. In some embodiments, the agent reversibly tethers the target RNA to the matrix embedding the biological sample. In some embodiments, the kit further comprises an agent for removing the embedded biological sample from the matrix. In some embodiments, the agent for removing the embedded biological sample comprises a clearing agent capable of clearing at least a subset of proteins and/or lipids in the biological sample. In some embodiments, the kit further comprises an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. [0399] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for tethering a target RNA of the biological sample to the matrix; an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises a first probe and a second probe, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA and the second probe comprises a sequence substantially complementary to a second portion of the target RNA and a capture sequence that is configured to be hybridized or ligated to the capture domain of the capture probe. In some embodiments, the capture sequence is substantially complementary to the capture domain of the capture probe. In some embodiments, the kit further comprises an agent for ligating together the first probe and the second probe hybridized to the target RNA tethered to the hydrogel matrix, thereby allowing for generation of a ligation product that is a proxy of the target RNA. In some embodiments, the kit further comprises an agent for releasing the ligation product from the hydrogel matrix, thereby allowing for hybridization or ligation of the released ligation product to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises a splint oligonucleotide that hybridizes to a portion of the released ligation product and a portion of the capture domain,^ny-2785575 202412020440 thereby allowing ligation using the splint oligonucleotide as a template. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the ligation product, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a sequencing primer and/or a sequencing adapter. In some embodiments, the sequencing primer can hybridize to the functional sequence or a complement thereof. In some embodiments, the sequencing adapter can be ligated to the functional sequence or a complement thereof, or can be ligated to the capture sequence or a complement thereof. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. [0400] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for tethering a target RNA of the biological sample to the matrix; an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises an agent for releasing the target RNA from the hydrogel matrix and thereby allowing for hybridization or ligation of the released target RNA to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises a splint oligonucleotide that hybridizes to a portion of the released target RNA and a portion of the capture domain, thereby allowing ligation using the splint oligonucleotide as a template. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a sequencing primer and/or a sequencing adapter. In some embodiments, the sequencing primer can hybridize to a sequence of the target RNA or a complement thereof. In some embodiments, the sequencing adapter can be ligated to a sequence of the target RNA or a complement thereof. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. [0401] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for tethering a^ny-2785575 202412020440 target RNA of the biological sample to the matrix; an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises a probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix. In some embodiments, the kit further comprises an agent for extending the probe, thereby appending a homopolymer sequence at the 3’ end of the extended probe. In some embodiments, the kit further comprises an agent for releasing the extended probe from the hydrogel matrix, thereby allowing for hybridization or ligation of the released extended probe to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises a splint oligonucleotide that hybridizes to a portion of the released extended probe and a portion of the capture domain, thereby allowing ligation using the splint oligonucleotide as a template. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a sequencing primer and/or a sequencing adapter. In some embodiments, the sequencing primer can hybridize to a sequence of the released extended probe or a complement thereof. In some embodiments, the sequencing adapter can be ligated to a sequence of the released extended probe or a complement thereof. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. [0402] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for tethering a target RNA of the biological sample to the matrix; an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises a first probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix. In some embodiments, the kit further comprises an agent for extending the first probe, thereby appending a homopolymer sequence at the 3’ end of the extended probe. In some embodiments, the kit further comprises a second probe for hybridizing to the extended probe in the hydrogel matrix and further extending the extended probe using the second probe as a template, wherein the extension comprises incorporating a polyuridine-^ny-2785575 202412020440 polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe. In some embodiments, the kit further comprises an agent for releasing the further extended probe from the hydrogel matrix and thereby allowing for hybridization or ligation of the released further extended probe to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises a splint oligonucleotide that hybridizes to a portion of the released further extended probe and a portion of the capture domain, thereby allowing ligation using the splint oligonucleotide as a template. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the further extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a sequencing primer and/or a sequencing adapter. In some embodiments, the sequencing primer can hybridize to a sequence of the further extended probe or a complement thereof. In some embodiments, the sequencing adapter can be ligated to a sequence of the further extended probe or a complement thereof. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. [0403] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence configured to be hybridized or ligated to the capture domain, (ii) a linker, and (iii) a reactive group, and wherein the transposome of the plurality is for tagmenting an open chromatin in the biological sample, and wherein the reactive group is for tethering the tagmented open chromatin to the hydrogel matrix. In some embodiments, the kit further comprises an agent for releasing tagmented open chromatin from the hydrogel matrix, thereby allowing for hybridization or ligation of the released tagmented open chromatin to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the tagmented open chromatin or a complement thereof, and the sequence of the spatial barcode or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a^ny-2785575 202412020440 sequencing primer and/or a sequencing adapter. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. [0404] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample. In some embodiments, the kit further comprises a transposome-binding moiety complex for binding to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody-binding moiety, (iii) a transposon end sequence comprising a capture sequence that is complementary to the capture domain of the capture probe on the array, and (iv) a reactive group, wherein the transposome binding moiety complex is for tagmenting a genomic DNA in the biological sample and wherein the reactive group is for tethering the tagmented genomic DNA to the hydrogel matrix. In some embodiments, the kit further comprises an agent for releasing the tagmented genomic DNA from the hydrogel matrix, thereby allowing for hybridization or ligation of the released tagmented genomic DNA to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the tagmented genomic DNA or a complement thereof, and the sequence of the spatial barcode or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a sequencing primer and/or a sequencing adapter. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. [0405] In some embodiments, the kit comprises: a matrix-forming agent for embedding the biological sample in a matrix (e.g., a hydrogel matrix); an agent for removing the embedded biological sample from the matrix; and an array comprising a capture probe, the capture probe comprising a capture domain and a spatial barcode. In some embodiments, the kit further comprises an analyte capture agent (e.g., an antibody-oligonucleotide congjugate) comprising (i) a binding moiety (e.g., an antibody or epitope binding fragment thereof, aptamer, etc.) that binds to a target protein in the biological sample, (ii) a capture sequence that is^ny-2785575 202412020440 complementary to the capture domain of the capture probe on the array, (iii) a barcode sequence associated with the binding moiety, and (iv) a reactive group, wherein the reactive group is for tethering the analyte capture agent to the hydrogel matrix. In some embodiments, the kit further comprises an agent for releasing the analyte capture agent or a portion thereof (e.g., comprising at least a portion of the capture sequence and at least a portion of the barcode sequence) from the hydrogel matrix, thereby allowing for hybridizing or ligating of the capture sequence (or a portion thereof) of the released analyte capture agent or portion thereof to the capture domain of the capture probe on the array. In some embodiments, the kit further comprises an agent for determining the sequence of all or a portion of the barcode sequence associated with the binding moiety or a complement thereof, and the sequence of the spatial barcode or a complement thereof. In some embodiments, the agent for sequence determination is an agent for nucleic acid sequencing, such as a sequencing primer and/or a sequencing adapter. In some embodiments, the kit further comprises instructions for using one or more kit components for analyzing the biological sample, according to a method disclosed herein. IV. APPLICATIONS [0406] In some aspects, the provided embodiments can be applied in a method of analyzing nucleic acid sequences, such as a spatial transcriptomic analysis, for example from intact tissues or samples in which the spatial information has been preserved. In some aspects, the embodiments can be applied on a spatial array, e.g., for analysis at single cell resolution. In some aspects, the embodiments can be applied in multiplexed nucleic acid analysis. In some aspects, the embodiments can be applied for multiplexed nucleic acid and protein analysis. In some aspects, the provided embodiments can be used to identify or detect regions of interest in a biological sample or tissue sample by analyzing gene and/or protein expression, such as target RNAs and/or chromatin regions, as well as non-nucleic acid targets such as proteins and portions thereof. [0407] In some embodiments, the analytes in a sample to be analyzed using a kit or composition disclosed herein can comprise more than one nucleotides and/or one or more proteins of interest. In some embodiments, the analytes can comprisea single nucleotide of interest. In some embodiments, the analytes can comprise a single-nucleotide polymorphism (SNP). In some embodiments, the analytes can comprise a single-nucleotide variant (SNV). In some embodiments, the analytes can comprise a single-nucleotide substitution. In some 135^ny-2785575 202412020440 embodiments, the single nucleotide is a point mutation. In some embodiments, the single nucleotide is a single-nucleotide insertion. In some embodiments, the single nucleotide is a single-nucleotide deletion. In some embodiments, the analytes can comprise a plurality of nucleotides of interest, for example a “hot spot” comprising two or more nucleotide variants, mutants, insertions or deletions. In some embodiments, the analytes can comprise one or more proteins of interest. For example, the analytes can comprise one or more targeted proteins as identified by analyte capture agents. [0408] In some aspects, the embodiments can be applied in investigative and/or diagnostic applications, for example, for characterization or assessment of particular cell or a tissue from a subject. Applications of the provided method can comprise biomedical research and clinical diagnostics. For example, in biomedical research, applications comprise, but are not limited to, spatially resolved gene expression analysis and gene expression profiling for biological investigation or drug screening. In clinical diagnostics, applications comprise, but are not limited to, detecting gene markers such as disease, immune responses, bacterial or viral DNA/RNA for patient samples. [0409] In some aspects, the embodiments can be applied to visualize the distribution of genetically encoded markers in whole tissue at subcellular resolution, for example, chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of gene alleles indicative of a predisposition towards disease or good health, likelihood of responsiveness to therapy, or in personalized medicine or ancestry. EXAMPLES [0410] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure. Example 1: Spatial Transcriptomic Profiling of FFPE Tissue Sections Using Probe Ligation on Target RNA Tethered to a Hydrogel [0411] This example demonstrates spatial transcriptomic profiling of an FFPE tissue section mediated by tethering of the 3’ end of a target mRNA molecule to a hydrogel. In this scenario, the mRNA is indirectly detected by using an RNA templated ligation workflow. [0412] An FFPE tissue section on a slide is baked, dewaxed, and rehydrated. In some cases, the rehydrated sample is stained using a morphological stain such as H&E stain and 136^ny-2785575 202412020440 imaged, optionally using bright field microscopy (e.g., BF imaging). The sample may be destained as depicted in the workflow of FIG. 12A. Hydrogel formation is carried out by forming a hydrogel that is intermeshed with the tissue section, such that the hydrogel permeates the tissue section. During and/or after the formation of the tissue section intermeshed hydrogel (e.g., tissue section in hydrogel), RNA molecules from the tissue section are 3’ tethered or 5’ tethered to the hydrogel matrix, for instance by a cleavable linker. If used, the cleavable linker can comprise a disulfide bond. The RNA molecules are tethered to the hydrogel at their location, or in close proximity to their location, within the tissue section. [0413] The tissue section is decrosslinked before or after tethering of the target molecule (e.g., target RNA in this example) to the hydrogel matrix. A majority or most of the tissue section is removed by a clearing process to remove proteins (e.g., ribosome components), lipids, and other cellular components in the tissue section, leaving the target RNA tethered to the hydrogel matrix at the location to where it was present, or in proximity to where it was present, in the tissue section. [0414] An RNA templated ligation (RTL) probe pair is hybridized to the RNA tethered to the hydrogel matrix. A left-hand side (LHS) probe and a right-hand side (RHS) probe are hybridized to adjacent sequences of the tethered RNA as depicted in FIG. 12B. The LHS probe comprises a functional sequence (e.g., Read 2S) at its 5’ end and a sequence domain that is complementary to a target mRNA molecule at the 3’ end. The RHS probe comprises a capture sequence that is complementary to a capture domain of a capture probe on a spatial array, in this example a poly(A) sequence at its 3’ end and a sequence domain that is complementary to a target mRNA molecule at its 5’ end. The 3’ end of the LHS probe and the 5’ end of the RHS probe (e.g., the two probes of the RTL probe pair) can hybridize adjacently to the target mRNA or can be non-adjacent, followed by a gap fill extension to bring the two probes together. FIG. 12B demonstrates adjacent RTL probe hybridization on a target mRNA. Following probe hybridization, the two probes are ligated together to generate a ligation product that serves as a proxy of the target mRNA that is tethered to the hydrogel. One or more washes are performed during probe hybridization, and following ligation, the hydrogel matrix is washed one or more times. [0415] The ligation products can be released from the tethered target mRNA and migrated to an arrayed slide for capture by the capture domains of the capture probes on the^ny-2785575 202412020440 spatial array which also comprises a hydrogel. A capture probe of the hydrogel barcoded capture array comprises a functional sequence (e.g., Read 1 primer sequence), a spatial barcode sequence, a unique moleculear identifier or UMI sequence, and capture domain, in this case a poly(dT)VN sequence at the 3’ end. In this example, the capture domain in the capture probe captures the ligation product by hybridization of the poly(dT)VN sequence to the poly(A) domain of the ligation products of the RNA targets as depicted in FIGS. 12C. [0416] Following capture of ligated RTL probes, a spatially labeled polynucleotide is generated and sequenced to determine all or a portion of the RTL probe sequence or complement thereof (which is a proxy of the target RNA sequence) and all or a portion of the spatial barcode sequence or complement thereof (which provides the spatial information in the FFPE sample) in the same spatially labeled polynucleotide molecule. In this example, after hybridization, the capture probe and ligation product are extended, the extended ligation product is released from the spatial array and library preparation steps are performed in anticipation of downstream sequence analysis, which associates the spatial barcodes and target RNA sequences in the sequencing reads of the spatially labeled polynucleotides with the locations in the tissue sample, thereby identifying a spatial profile of RNA transcripts in the tissue sample. Example 2: Spatial Transcriptomic Profiling of FF Tissue Sections Mediated by 3’ or 5’ Target RNA Tethering to a Hydrogel and Capturing Released Target RNA [0417] This example shows spatial transcriptomic profiling of fresh frozen tissue sections mediated by 3’ target RNA tethering or 5’ target RNA tethering to a hydrogel. First, an FF tissue section is provided on a slide, stained using a morphological stain such as H&E stain, and imaged, optionally using bright field microscopy (e.g., BF imaging). The tissue section may be destained as depicted in the workflow of FIG. 13A. Hydrogel formation is carried out by forming a hydrogel that is intermeshed with the tissue section, such that the hydrogel permeates the tissue section. RNA molecules are 3’ tethered or 5’ tethered to the hydrogel matrix, for instance by a cleavable linker, during and/or after the formation of the hydrogel matrix. If used, the cleavable linker can comprise a disulfide bond. The tissue section embedded in the hydrogel is cleared to remove at least a portion of the proteins (e.g., ribosome components) and lipids, leaving the target RNA tethered to the hydrogel matrix. The slide holding the target RNA tethered to the hydrogel is inverted and aligned with a spatial array such that the hydrogel is sandwiched between the slide and the spatial array substrate. Tethered target RNAs are released, 138^ny-2785575 202412020440 for instance by cleaving the cleavable linkers or reversing the tethering by changing the pH of a boronic acid-based hydrogel that embeds the tissue section before the clearing step. In this example, the hybridization step during RNA capture occurs during the sandwich step, which also includes the release of the target RNA, prior to RNA capture. A capture probe of the hydrogel barcoded capture array comprises a functional sequence (e.g., Read 1 primer sequence), a spatial barcode sequence, a UMI sequence, and a poly(dT)VN sequence at the 3’ end. In this example, the capture probe captures the released target RNA by hybridization of the poly(dT)VN sequence of the capture domain of the capture probe to the poly(A) of the RNA target as depicted in FIGS. 13B. Following hybridization, the capture probe is extended via reverse transcription using the hybridized RNA as the template, for instance, as shown in FIG. 13B. A template switch oligo priming step followed by second strand synthesis are performed as depicted in FIG. 13C, to generate a spatially labeled polynucleotide, which is sequenced to determine all or a portion of the target RNA sequence or complement thereof and all or a portion of the spatial barcode sequence or complement thereof (which provides the spatial information in the sample) in the same spatially labeled polynucleotide molecule. [0418] Following target (e.g., RNA targets) release and capture, workflow proceeds with the step of amplifying material and generating libraries. Subsequent analysis is then performed associating the spatial barcodes and target RNA sequences in the sequencing reads of the spatially labeled polynucleotides with the locations in the tissue sample, thereby identifying a spatial profile of RNA transcripts (including spatial locations of RNA) in the tissue sample. Example 3: Spatial Transcriptomic Profiling of FF Tissue Sections Mediated by 3’ Target RNA Tethering to a Hydrogel and TSO-Based Capture [0419] This example shows spatial transcriptomic profiling of fresh frozen tissue sections comprising RNA tethering to a hydrogel. An overview of the workflow utilizing a 5’ RT-based gene expression analysis and TSO-based capture is depicted in FIG. 14. First, an FF tissue section is provided on a slide. The tissue section is H&E stained, imaged with a bright field microscope and optionally destained. Hydrogel formation is carried out by forming a hydrogel that is intermeshed with the tissue section, such that the hydrogel permeates the tissue section. Target RNA molecules are 3’ tethered to the hydrogel matrix, for instance, by a reversible/cleavable linker such as one comprising a disulfide bond. A clearing step (e.g., tissue removal) is performed leaving target RNA molecules tethered to the hydrogel matrix. Following 139^ny-2785575 202412020440 clearing, a reverse transcription step is performed on the gel tethered target RNA molecule, using a probe containing poly(dT) and non-poly(dT) sequences, of which the poly(dT) hybridizes to the tethered RNA. The probe is extended by reverse transcription using the hybridized tethered RNA as a template to incorporate the complement of the target RNA sequence into the extended probe. The reverse transcribed molecule that comprises a complement of the target RNA molecule can hybridize to the capture domain of the capture probe immobilized on the spatial array. Template switch oligonucleotide priming is performed followed by extension and incorporation of the complement of the capture probe domains into the reverse transcribed and hybridized molecule that comprises the complement of the RNA target. [0420] In some instances, the extended probe comprises a poly(C) sequence at the 3’ end. The extended probe and the target RNA hybridized thereto (which form a partially double stranded complex comprising a 3’ poly(C) overhang and a 3’ poly(A) overhang) can be released from the hydrogel matrix without digesting the target RNA (e.g., using an RNase H), such that the released partially double stranded complex can be captured by the 3’ poly(rG) sequence of a capture probe on a hydrogel barcoded capture array. Alternatively, in instances where the target RNA is digested and only the extended probe is captured, the capture probe can be extended using the poly(rG) sequence as a primer and the extended probe as a template to generate an extended capture probe that incorporates the target RNA sequence and a 3’ poly(A) sequence. [0421] In some instances, following template switch, the extended probe can be further extended by using the poly(C) sequence as a primer and the capture probe as a template to generate a further extended probe that appends the complement of the Read 1 primer sequence, spatial barcode, UMI, and TSO sequences to the complement of the target RNA sequence, thereby generating a spatially labeled polynucleotide (e.g., a cDNA molecule). After that, amplification of the spatially labeled polynucleotide and library preparation are performed. Subsequent analysis is performed associating the spatial barcode with the RNA target from the tissue section, thereby identifying the locations of the target RNA molecules in the tissue section, and the method can be used to identify spatial profiles of multiple different RNA transcripts in the tissue section.^ny-2785575 202412020440 Example 4: Spatial Transcriptomic Profiling of FF Tissue Sections Mediated by 3’ Target RNA Tethering to a Hydrogel and Poly(dT)-Based Capture [0422] This example shows spatial transcriptomic profiling of fresh frozen tissue sections comprising RNA tethering to a hydrogel. An overview of the workflow, utilizing a 5’ RT-based gene expression analysis and poly(dT)-based capture is depicted in FIG. 15. First, an FF tissue section is provided on a slide, H&E stained, imaged with a bright field microscope and optionally destained. A hydrogel matrix is then formed within the tissue section. Target RNA molecules are 3’ tethered to the hydrogel matrix, for instance, by a reversible/cleavable linker such as one comprising a disulfide bond. Tissue removal (e.g., using a clearing step) is performed leaving target RNA molecules tethered to the hydrogel matrix. Following clearing, a reverse transcription step is performed on the gel tethered target RNA molecule, using a probe containing poly(dT) and non-poly(dT) sequences. As shown in FIG. 15, the probe is extended by reverse transcription to incorporate the complement of the target RNA sequence into the extended probe which also comprises a poly(C) sequence at the 3’ end. A template switch oligonucleotide comprising a 5’ poly(U) sequence and a 3’ poly(rG) sequence is hybridized to the partially double stranded complex formed between the extended probe and the target RNA hybridized thereto, where the 3’ poly(rG) sequence hybridizes to the 3’ poly(C) overhang. Following this TSO priming step in the hydrogel, the extended probe is further extended using the poly(C) sequence as a primer and the template switch oligonucleotide as a template to generate a further extended probe that appends a 3’ poly(A) sequence to the extended probe comprising a complement of the target RNA sequence, thereby generating a cDNA molecule comprising a 3’ poly(A) sequence. The cDNA molecule can be released in a RNase H treatment step involving forming a hydrogel/hydrogel sandwich. In the releasing step, the RNase H degrades the RNA sequence of a DNA:RNA hybrid, thereby releasing the cDNA molecule. In the sandwiching step, the cDNA molecule (of the target RNA) comprising a 3’ poly(A) is captured via hybridization of the poly(A) by the capture probe’s poly(dT) sequence, and a spatially labeled polynucleotide can be generated by combining the target RNA/cDNA sequence with the spatial barcode sequence (or a complement thereof) in the same molecule. [0423] Following target release and capture, workflow proceeds with the step of amplifying material and generating libraries. For instance, second strand synthesis (to generate a spatially labeled polynucleotide) followed by amplification and library preparation are^ny-2785575 202412020440 performed. Subsequent analysis is performed associating the spatial barcode with the RNA target from the tissue section, thereby identifying the locations of the target RNA molecules in the tissue section, and the method can be used to identify spatial profiles of multiple different RNA transcripts in the tissue section. Example 5: Method for Combined Spatial ATAC-seq and Gene Expression Profiling using RNA-seq Mediated by Target Tethering in a Hydrogel [0424] Simultaneous profiling of transposase-accessible chromatin regions and gene expression in a biological sample can provide valuable insights into the mechanisms of gene regulation in living systems. Such mechanisms include, but are not limited to, mechanisms of organism development and mechanisms of disease development. [0425] Target molecules (e.g., transposed DNA and RNA transcripts and/or probes hybridized to the RNA transcripts) are tethered (e.g., via reversible tethering) to a hydrogel matrix. Tethered transposed DNA is used for the spatial ATAC-seq modality and tethered RNA transcripts and/or probes that hybridize to the RNA are used for the RNA-seq modality. In this example, transposase-accessible chromatin regions and transcripts are released and captured in a single sandwiching step on an array, and each array feature (e.g., an array spot comprises capture probes with the same spatial barcode) can contain different capture regions for capturing the target molecules for the spatial ATAC-seq modality and for the RNA-seq modality. This strategy combines 3’ or 5’ transcript tethering with a transposome assembly comprising a modified oligonucleotide with a reactive group (e.g., an an acrydite such as 5’-acrydite) for tethering of transposed DNA to a hydrogel matrix and a cleavable linker for controlled release of the transposed DNA from the matrix. After target tethering, the tissue is removed (e.g., by a clearing method), and the matrix with tethered transposed DNA and tethered RNA is sandwiched with a barcoded spatial array (e.g., an hydrogel spatial array) to control release and simultaneous capture of the target molecules. [0426] An exemplary workflow is depicted in FIG. 16A. The workflow comprises the following steps: fixing the FF tissue section; optional H&E/IF staining, imaging, and destaining; optional permeabilization of the biological sample; preparing and activating the RNA target for 3’ or 5’ tethering; transposition (e.g., by Tn5); hydrogel embedding; optionally descrosslinking the tissue embedded in the hydrogel; removing the tissue (e.g., clearing); and simultaneously releasing the target molecules from the sample hydrogel and capturing with the 142^ny-2785575 202412020440 hydrogel barcoded spatial capture array. The permeabilization step is carried out gently to facilitate the RNA target preparation step. After the clearing step the target molecules remain in the hydrogel, and sample proteins, organelles, and the like are washed away. As shown in FIGS. 16B-16D, a Tn5 transposome used in this example can comprise a first DNA adapter sequence comprising a capture sequence or a partial capture sequence that is configured to be hybridized or ligated (e.g., by using a splint oligonucleotide) to a capture domain of a capture probe on a spatial array, and a second DNA adapter sequence comprising a reactive group (e.g., 5’- acrydite), optionally the reactive group is connected to the second DNA adapter sequence via a cleavable linker. A modified design for the transposon in the Tn5 assembly is depicted in FIG. 16B. The modified transposon comprises a PCR handle (pR2), a disulfide linker and a 5’- acrydite at its terminus. The acrydite functionality is a reactive group for hydrogel embedding. The disulfide linker allows for chemically triggered release of the tethered target molecule (e.g., the transposed DNA). Components of the transposome are depicted in FIG. 16C. The structure of the transposed DNA is depicted in FIG. 16D. The acrydite group allows for tethering to the sample hydrogel. [0427] The target releasing step comprises contacting the sample hydrogel (e.g., tissue hydrogel on the sample substrate) with the hydrogel barcoded capture array (e.g., array of capture probes on the spatial array substrate), e.g., by sandwiching the hydrogel matrix containing target molecules tethered thereto between the sample substrate and the array substrate. Tethered targets are released by cleaving a cleavable linker, which can comprise a disulfide bond that is chemically cleavable. [0428] The structures of capture probes on the hydrogel barcoded array are shown in FIGS. 16E-16F. In this example, the capture probes comprise a mix of two capture domains specific to the two target nucleic acid modalities. A molecule comprising transposed DNA for the ATAC-seq modality is captured (e.g., by hybridization or ligation to the partial capture sequence) by the capture probe comprising the spacer shown in FIG. 16E. A molecule comprising a target RNA sequence or complement thereof for the RNA-seq modality is captured (e.g., via a poly(A) sequence) by the capture probe comprising a poly(dT) sequence shown in FIG. 16F. [0429] Following target release and capture, workflow proceeds with the step of amplifying material and generating libraries. Subsequent analysis is then performed to associate^ny-2785575 202412020440 the spatial barcodes and target RNA sequences, and associate the spatial barcodes and transposed DNA from open chromatin, in the sequencing reads with the locations in the tissue section, thereby identifying a spatial profile of open chromatin and RNA transcripts in the tissue section. Example 6: Method for Combined Spatial ChIP-seq and Gene Expression Profiling using RNA-seq Mediated by Target Tethering in a Hydrogel [0430] ChIP-seq provides information on genome-wide DNA binding sites and associated DNA-binding proteins (e.g., transcription factors/co-factors) as well as the localization of histones and their modifications throughout the genome. Combined with gene expression profiling, ChIP-seq represents a powerful tool to investigate the mechanism of gene regulation. [0431] This example shows the simultaneous release and capture of i) target molecules containing sequence information of genomic domains comprising an epigenetic modification (e.g., DNA modification such as methylation or histone modification such as acetylation) or bound by a protein (e.g., a DNA binding protein such as a transcription factor) along with ii) target molecules containing sequence information of RNA transcripts from a cell or tissue sample. The target molecules are immobilized (e.g., reversibly tethered) to a hydrogel matrix followed by sample removal (e.g., by clearing). The target molecules are then captured by capture probes on a spatial array for combined analysis of the ChIP-seq modality and the RNA-seq modality. [0432] An example of a combined spatial ChIP-seq and transcriptomic profiling workflow is provided in FIG. 17A. An FF tissue section is subjected to the following steps: fixing; optional H&E/IF staining, imaging, and destaining; optional gentle permeabilization of the sample to facilitate the next steps; preparing RNA in the sample for 3’ or 5’ tethering; incubating with the primary and secondary antibodies; transposition (e.g., using pA-Tn5); embedding the sample within a hydrogel; optionally descrosslinking the sample embedded in the hydrogel; and removing the tissue (e.g., clearing), leaving the target molecules tethered to the hydrogel. The target release step comprises contacting the sample hydrogel (e.g., tissue hydrogel on the sample substrate) with the hydrogel barcoded spatial capture array (e.g., array of capture probes on the array substrate), e.g., by sandwiching the hydrogel matrix containing target molecules tethered thereto between the sample substrate and the array substrate. Tethered targets^ny-2785575 202412020440 are released by cleaving a cleavable linker, which can comprise a disulfide bond that is chemically cleavable by a reducing agent such as DTT. [0433] As shown in FIGS. 17B-17D, transposition in a method in this example can involve tagmentation and cleavage of the target molecules using: a primary antibody that binds to a target modification (e.g., of DNA or histone) or a target DNA-binding protein; a secondary antibody that binds to the primary antibody; and a transposome-binding moiety complex comprising protein A (pA)-Tn5 transposome complex with DNA adapter sequences. The transposome-binding moiety complex is targeted to the target modification or target DNA- binding protein, tagments at the chromatin close to the target modification or target DNA- binding protein binding site, and appends the DNA adapter sequences to transposed DNA from the chromatin. One of the DNA adapter sequences can comprise a capture sequence or a partial capture sequence that is configured to be hybridized or ligated (e.g., by using a splint oligonucleotide) to a capture domain of a capture probe on a spatial array, and another one of of the DNA adapter sequences can comprise a reactive group (e.g., 5’-acrydite), optionally the reactive group is connected to the DNA adapter sequence via a cleavable linker. The DNA adapter sequences can be in modified transposon ends and are depicted in FIG. 17C together with a pA-Tn5 transposase complex. [0434] The target releasing step comprises contacting the sample hydrogel (e.g., tissue hydrogel on the sample substrate) with the hydrogel barcoded capture array (e.g., array of capture probes on the array substrate), e.g., by sandwiching the hydrogel matrix containing target molecules tethered thereto between the sample substrate and the array substrate. Tethered targets are released by cleaving a cleavable linker, which can comprise a disulfide bond that is chemically cleavable. A molecule comprising transposed DNA for the ChIP-seq modality is captured (e.g., by hybridization or ligation to the partial capture sequence) by a capture probe comprising a spacer (e.g., as shown in FIG. 19, middle panel), and a molecule comprising a target RNA sequence or complement thereof for the RNA-seq modality is captured (e.g., via a poly(A) sequence) by a capture probe comprising a poly(dT) or a poly(rG) sequence (e.g., as shown in FIG. 19, lower panel). [0435] Following target release and capture, workflow proceeds with the step of amplifying material and generating libraries. Subsequent analysis is then performed to associate the spatial barcodes and target RNA sequences, and associate the spatial barcodes and transposed^ny-2785575 202412020440 DNA from chromatin with a chromatin modification and/or a protein binding region of interest, in the sequencing reads with the locations in the tissue sample, thereby identifying a spatial profile of chromatin modification and/or protein binding and RNA transcripts in the tissue sample. Example 7: Method for Combined Spatial Protein and Gene Expression Profiling Mediated by Target Tethering in a Hydrogel [0436] This example shows the release and capture of i) target molecules containing identifying information (e.g., barcode sequences) of proteins (or other non-nucleic acid analytes) from a cell or tissue sample, along with ii) target molecules containing sequence information of RNA transcripts from the cell or tissue sample. The target molecules are immobilized (e.g., reversibly tethered) to a hydrogel matrix followed by sample removal (e.g., by clearing). The target molecules are then captured by capture probes on a spatial array for combined analysis of the proteins (or other non-nucleic acid analytes) and the RNA transcripts. [0437] An example of a combined spatial protein and transcriptomic profiling workflow is provided in FIG. 18A. An FFPE tissue section is subjected to the following steps: baking the tissue section; dewaxing; rehydrating; optional H&E staining, bright field imaging, and destaining; hydrogel embedding of the sample and tethering RNA in the sample by 3’ or 5’ tethering to hydrogel; descrosslinking the tissue in the sample hydrogel; RTL probe hybridizing; RTL probe ligating; incubating the hydrogel embedded sample with antibody-oligonucleotide conjugates for proteins (e.g., analyte capture agents comprising protein binding moieties and associated oligonucleotides), and tethering the oligonucleotides of the antibody-oligonucleotide conjugates to the hydrogel matrix; and removing the tissue (e.g., clearing), leaving the target molecules (e.g., ligated RTL probes, and oligonucleotides of the antibody-oligonucleotide conjugates comprising identifying information (e.g., antibody barcodes) for the proteins) tethered to the hydrogel. An antibody-oligonucleotide conjugate used in this example is depicted in FIG. 18B. The 5’ end of the oligonucleotide includes an acrydite group for tethering to the hydrogel. On the 3’ end, as depicted in FIG. 18B, the antibody-oligonucleotide conjugate includes a capture domain sequence that is complementary to a capture domain of a capture probe on a hydrogel barcoded spatial capture array. Additionally, the antibody-oligonucleotide conjugate comprises a PCR handle (pR2), a conjugated antibody (Ab) and an antibody-specific barcode that identifies the conjugated antibody and therefore the protein to which it is bound. Finally, to 146^ny-2785575 202412020440 allow for reversible tethering, a cleavable linker composed of a disulfide bond is incorporated near the acrydite functionality. The antibody targeting the protein of interest in the biological sample is digested during the tissue clearing/digestion step, whereas the oligonucleotide tag is held in place until the release step. In this example, the release is triggered by chemically cleaving the disulfide bond of the cleavable linker. Alternative antibody-oligonucleotide conjugates are shown in FIGS. 18C-18D. [0438] The target release step comprises contacting the sample hydrogel (e.g., tissue hydrogel on the sample substrate) with the hydrogel barcoded capture array (e.g., array of capture probes on the array substrate), e.g., by sandwiching the hydrogel matrix containing target molecules tethered thereto between the sample substrate and the array substrate. Tethered analyte capture agents can be released by cleaving a cleavable linker (e.g., as shown in any one of FIGS. 18B-18D), which can comprise a disulfide bond that is chemically cleavable by a reducing agent such as DTT. Tethered RNA transcripts and ligated RTL probes can be released essentially as described in Example 1. [0439] A molecule comprising identifying information (e.g., an antibody-specific barcode sequence) of a protein is captured by a capture probe comprising a capture sequence that is hybridized or ligated to the capture sequence in an analyte capture agent, e.g., as shown in FIGS. 18E-18F and FIG. 19 (upper panel). A molecule comprising a target RNA sequence or complement thereof for the RNA-seq modality is captured (e.g., via a poly(A) sequence) by a capture probe comprising a poly(dT) or a poly(rG) sequence (e.g., as shown in FIG. 19, lower panel). [0440] Following target release and capture, workflow proceeds with the step of probe elongating, amplifying material and generating libraries. Subsequent analysis is then performed to associate the spatial barcodes and target RNA sequences, and associate the spatial barcodes and protein-identifying barcodes, in the sequencing reads with the locations in the tissue sample, thereby identifying a spatial profile of proteins and RNA transcripts in the tissue sample. Example 8: Reversible Tethering of mRNA to Boronic Acid Hydrogels [0441] In this example, boronic acid moieties are embedded within the sample hydrogel matrix to passively capture 3’ RNA ends that have been polished. In this approach, the tethering of the RNA target molecules occurs if the pH of the system is greater than the pKa of 147^ny-2785575 202412020440 boronic acid, due to the formation of boronate esters between the boronic acid moieties of the hydrogel matrix with the 3’ RNA diols of the target RNA molecule. A pH below the pKa reverses the tethering. In this example, no separate tether linker molecule is needed and the tethering can be reversed by changing the pH of the system. [0442] Two different hydrogels are prepared: a boronic acid hydrogel (e.g., sample hydrogel) and a standard hydrogel (e.g., non-reversible hydrogel, non-boronic acid hydrogel, array hydrogel, or reader hydrogel). Capture probes of thespatial array are intermeshed in the standard hydrogel. The sandwich arrangement including the boronic acid hydrogel-based reversible tethering scheme is seen in FIG. 20A. [0443] A boronic acid hydrogel is formed by intermeshing with the tissue section and the hydrogel permeating the tissue section, and RNA molecules in the tissue section can be tethered to the boronic acid hydrogel via the 3’ RNA diols. A clearing step is used to remove unwanted cellular components such as ribosomes and prepare the sample to interface with the (non-boronic acid) hydrogel containing the patterned spatial array. [0444] The sample hydrogel and the standard hydrogel are sandwiched between the sample subtrate and the spatial array substrate. A drop in pH triggers release of the target mRNA, followed by diffusion of the mRNA target to the capture sptial array. Specifically, in the presence of a slightly acidic buffer, the boronate ester bonds are reversed, thereby allowing the RNA molecules to be released from the boronic acid hydrogel for migration to the capture probes in the spatial array hydrogel. The proximity of the spatial array oligonucleotides helps limit lateral diffusion of the released RNA molecules through the sample hydrogel and the spatial array hydrogel to mitigate mis-localization. [0445] Capacitance can be utilized to limit lateral diffusion and facilitate migration in a direction toward the spatial capture array, thereby increasing the efficiency of the nucleic acid capture process. The external field driving the electrophoretic migration of the target mRNA to the spatial capture array is provided by thin metal plates (e.g., copper plates) connected to a power source, as depicted in FIG. 20B, where each plate acts as a negatively charged cathode and a positively charged anode, respectively. [0446] Thin conductive layers applied to both the sample substrate and the spatial capture array substrate can be used to provide for the capacitance. FIG. 20C depicts a thin film of indium tin oxide (ITO) deposited on both the sample substrate and the spatial capture array^ny-2785575 202412020440 substrate. Here, the spatial capture array gel and the boronic acid hydrogel are deposited on a methacrylic acid coated oxide surface. ITO readily conducts current allowing for the ability to actively electrophorese (e.g., to cause migration by electrophoresis) the untethered (e.g., released) RNA onto the spatial capture array substrate. ITO coated substrates allow for a greater degree of control in the release and electrophoretic driven migration of nucleic acids (e.g., directionally directed, or anisotropic diffusion), as voltage, amperage, ionic conditions in the buffer and temperature are optimized and controlled. [0447] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.^ny-2785575

Claims

202412020440 CLAIMS 1. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a matrix; b) tethering a target nucleic acid in the biological sample to the matrix; c) removing a subset of cellular components from the biological sample in the matrix; d) releasing the target nucleic acid from the matrix; e) hybridizing or ligating the released target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and f) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. 2. The method of claim 1, wherein the target nucleic acid comprises DNA and/or RNA. 3. The method of claim 2, wherein the RNA is mRNA. 4. The method of any one of claims 1-3, wherein the matrix is a hydrogel matrix. 5. The method of any one of claims 1-4, wherein the tethering comprises tethering a 3’ end of the target nucleic acid to the matrix. 6. The method of any one of claims 1-4, wherein the tethering comprises tethering a 5’ end of the target nucleic acid to the matrix. 7. The method of any one of claims 1-6, wherein the tethering comprises enzymatic tethering. 8. The method of any one of claims 1-7, wherein the tethering comprises non-enzymatic tethering. 9. The method of any one of claims 1-8, wherein the tethering comprises a periodate oxidation reaction or a reaction with boronic acid. 10. The method of any one of claims 1-9, wherein the tethering comprises tethering the target nucleic acid to the matrix via a linker.^ny-2785575 202412020440 11. The method of any one of claims 1-10, wherein the linker comprises a cleavable linker. 12. The method of claim 11, wherein the cleavable linker comprises a disulfide bond. 13. The method of any one of claims 1-12, wherein the matrix is formed using N,N′- Bis(acryloyl)cystamine (BAC) as a crosslinker. 14. The method of any one of claims 1-13, wherein the removing of the subset of cellular components from the biological sample comprises removing at least a subset of ribosomes from the biological sample. 15. The method of any one of claims 1-14, wherein the releasing comprises contacting the matrix with a chemical agent configured to cleave a cleavable linker between the target nucleic acid and the matrix. 16. The method of any one of claims 1-14, wherein the releasing comprises illuminating the matrix with light, thereby cleaving a cleavable linker between the target nucleic acid and the matrix. 17. The method of any one of claims 1-14, wherein the releasing comprises altering the pH of a solution in contact with the matrix, thereby releasing the tethered target nucleic acid. 18. The method of claim 17, wherein the target nucleic acid is tethered to the matrix via a boronate ester bond. 19. The method of claim 18, wherein the boronate ester bond is formed between a boronic acid moiety and 3’ diols of the target nucleic acid, wherein the target nucleic acid is RNA. 20. The method of any one of claims 1-19, wherein the matrix comprises a boronic acid- based hydrogel matrix. 21. The method of any one of claims 1-20, wherein the releasing comprises exposing the matrix to a temperature of about 40°C to about 75°C, thereby releasing the tethered target nucleic acid, optionally wherein the matrix is a hydrogel matrix comprising poly(N- isopropylacrylamide). 22. The method of any one of claims 1-21, wherein the target nucleic acid comprises a cellular nucleic acid in the biological sample, a product of the cellular nucleic acid generated in^ny-2785575 202412020440 the biological sample, a nucleic acid probe that directly or indirectly binds to the cellular nucleic acid, or a product of the nucleic acid probe generated in the biological sample. 23. The method of any one of claims 1-22, wherein the matrix and the biological sample embedded therein are on a first solid support, and the spatial array is on a second solid support. 24. The method of any one of claims 1-23, wherein the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, is determined using nucleic acid sequencing. 25. The method of claim 24, wherein the nucleic acid sequencing comprises sequencing by synthesis, sequencing by ligation, sequencing by binding, sequencing by avidity, sequencing by hybridization, or any combination thereof. 26. The method of any one of claims 1-25, further comprising directing the released target nucleic acid to the capture probe. 27. The method of claim 26, wherein the directing comprises directing via a capacitor, thereby generating an electrophoretic migration of the released target nucleic acid from the matrix to the spatial array. 28. The method of claim 27, wherein at least one plate of the capacitor comprises a coating of indium tin oxide. 29. The method of any one of claims 1-28, wherein the matrix is a first hydrogel matrix and the capture probe is affixed on the spatial array and embedded in a second hydrogel matrix. 30. The method of claim 29, wherein the first hydrogel matrix and the second hydrogel matrix are of the same composition. 31. The method of claim 29, wherein the first hydrogel matrix and the second hydrogel matrix are of different compositions. 32. The method of any one of claims 1-31, comprising determining a spatial gene expression profile of the target nucleic acid in the biological sample.^ny-2785575 202412020440 33. The method of any one of claims 1-32, wherein a plurality of different target nucleic acids are tethered to the matrix in a), released from the matrix in c), and hybridized or ligated to capture domains of capture probes on the spatial array. 34. The method of any one of claims 1-33, comprising digesting and/or clearing the embedded biological sample from the matrix. 35. The method of any one of claims 1-34, wherein the biological sample is a fresh tissue sample, a frozen tissue sample, or a fixed tissue sample. 36. The method of any one of claims 1-35, wherein the biological sample is a fresh frozen tissue section or a formalin-fixed paraffin-embedded tissue section. 37. The method of any one of claims 1-36, comprising fixing the biological sample prior to embedding in the matrix, and decrosslinking the embedded biological sample prior to or during the removing of the subset of cellular components. 38. A method for analyzing a biological sample, the method comprising: a) contacting a biological sample comprising a target nucleic acid with an attachment agent comprising a boronic acid moiety capable of covalently reacting with at least one 2’,3’ vicinal diol of the target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent in the biological sample, wherein the biological sample and the attachment agent are contacted under conditions suitable to form a covalent bond between the boronic acid moiety and the 2’,3’ vicinal diol of the target nucleic acid; b) contacting the biological sample with the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the target nucleic acid to the matrix; c) clearing the embedded biological sample from the matrix; d) releasing the target nucleic acid from the matrix; e) capturing the target nucleic acid by a capture probe on a spatial array, wherein the capture probe comprises a capture domain and a spatial barcode; and f) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof. 39. A method for analyzing a biological sample, the method comprising:^ny-2785575 202412020440 a) contacting the biological sample comprising a fragmented target nucleic acid with a 3’ phosphatase to provide a fragmented target nucleic acid comprising a 2’,3’-vicinal diol moiety; b) contacting the biological sample with a formylation reagent, wherein the formylation reagent converts the 2’,3’-vicinal diol moiety into a 2’,3’-dialdehyde moiety, optionally wherein the formylation reagent comprises sodium (meta)periodate; c) contacting the biological sample with an attachment agent and a matrix-forming agent, the attachment agent comprising at least one aldehyde-reactive group capable of forming a covalent bond with at least one aldehyde of the 2’,3’-dialdehyde moiety of the fragmented target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the fragmented target nucleic acid to the matrix; d) clearing the embedded biological sample from the matrix; e) releasing the fragmented target nucleic acid from the matrix; f) hybridizing the fragmented target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and g) determining the sequence of all or a portion of the fragmented target nucleic acid, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof. 40. The method of any one of claims 1-39, wherein the capture probe further comprises a cleavage domain, a functional domain, a unique molecular identifier, or a combination thereof. 41. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) applying a first probe and a second probe to the hydrogel matrix, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA and the second probe comprises a sequence substantially complementary to a second portion of the target RNA and a capture sequence that is configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode;^ny-2785575 202412020440 d) hybridizing the first probe and the second probe to the target RNA tethered to the hydrogel matrix; e) ligating together the first probe and the second probe hybridized to the target RNA tethered to the hydrogel matrix, thereby generating a ligation product that is a proxy of the target RNA; f) releasing the ligation product from the hydrogel matrix and hybridizing or ligating the released ligation product to the capture domain of the capture probe on the spatial array; and g) determining a spatial transcriptomic profile for the biological sample by determining a sequence of all or a portion of the ligation product, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof. 42. The method of claim 41, wherein prior to a), the biological sample is a crosslinked biological sample, and wherein in a), the crosslinked biological sample is embedded in the hydrogel matrix and the target RNA in the crosslinked biological sample is tethered to the hydrogel matrix. 43. The method of claim 42, wherein between a) and b), the method comprises decrosslinking the crosslinked biological sample embedded in the hydrogel matrix. 44. The method of any one of claims 41-43, wherein prior to a), the biological sample is a fixed tissue sample. 45. The method of any one of claims 41-44, wherein prior to a), the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue section. 46. The method of any one of claims 41-45, wherein the target RNA is 3’ tethered to the hydrogel matrix. 47. The method of any one of claims 41-45, wherein the target RNA is 5’ tethered to the hydrogel matrix. 48. The method of any one of claims 41-47, wherein the functional sequence in the first probe is in a 5’ overhang upon hybridization of the first probe to the target RNA. 49. The method of any one of claims 41-48, wherein the functional sequence in the first probe comprises a primer binding sequence or a complement thereof.^ny-2785575 202412020440 50. The method of any one of claims 41-49, wherein the capture sequence in the second probe is in a 3’ overhang upon hybridization of the second probe to the target RNA. 51. The method of any one of claims 41-50, wherein the capture sequence in the second probe comprises a 3’ polyadenine sequence. 52. The method of any one of claims 41-51, wherein the first probe and the second probe are ligated using the target RNA as a template without gap filling prior to the ligation. 53. The method of any one of claims 41-51, wherein the first probe and the second probe are ligated using the target RNA as a template with gap filling prior to the ligation, wherein the gap filling comprises hybridization of a gap-fill oligonucleotide to the target RNA and/or primer extension of the first probe or the second probe by a polymerase. 54. The method of any one of claims 41-53, wherein the releasing in f) comprises releasing the tethered RNA from the hydrogel matrix. 55. The method of any one of claims 41-54, wherein the releasing in f) comprises releasing the ligation product from the target RNA. 56. The method of any one of claims 41-55, wherein the releasing in f) comprises contacting the hydrogel matrix with an RNase H. 57. The method of any one of claims 41-56, wherein the capture domain in the capture probe comprises a 3’ poly(dT) sequence. 58. The method of any one of claims 41-57, wherein between f) and g), the method comprises generating a spatially labeled polynucleotide comprising (i) from 5’ to 3’, a sequence of the released ligation product and a complementary sequence of the spatial barcode, or (ii) from 5’ to 3’, a sequence of the spatial barcode and a complementary sequence of the released ligation product. 59. The method of claim 58, wherein the spatially labeled polynucleotide or a portion thereof is released from the spatial array for analysis. 60. The method of claim 59, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing.^ny-2785575 202412020440 61. The method of any one of claims 41-60, wherein the method comprises imaging the spatial array using fluorescence microscopy. 62. The method of any one of claims 41-61, wherein the method comprises amplifying the released ligation product or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released ligation product or the spatially labeled polynucleotide or an amplification product thereof. 63. The method of any one of claims 41-62, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array. 64. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) releasing the target RNA from the hydrogel matrix; d) hybridizing or ligating the released target RNA to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and e) determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof. 65. The method of claim 64, wherein prior to a), the biological sample is a fresh frozen tissue sample or a decrosslinked tissue sample, or wherein prior to a), the biological sample is a fixed tissue sample, and between a) and b), the biological sample embedded in the hydrogel matrix is decrosslinked. 66. The method of claim 64 or claim 65, wherein the target RNA is 3’ tethered to the hydrogel matrix. 67. The method of claim 64 or claim 65, wherein the target RNA is 5’ tethered to the hydrogel matrix. 68. The method of any one of claims 64-67, wherein the capture domain in the capture probe comprises a 3’ poly(dT) sequence.^ny-2785575 202412020440 69. The method of any one of claims 64-68, wherein between d) and e), the method comprises generating an extended capture probe comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released target RNA. 70. The method of claim 69, wherein the extended capture probe further comprises a 3’ end homopolymer sequence. 71. The method of claim 69 or claim 70, further comprising hybridizing a template switch oligonucleotide to the extended capture probe, and extending the extended capture probe using the template switch oligonucleotide as a template to generate a further extended capture probe. 72. The method of claim 71, further comprising hybridizing a primer to the further extended capture probe at a sequence complementary to the template switch oligonucleotide, and extending the primer using the further extended capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the released target RNA and a complementary sequence of the spatial barcode. 73. The method of claim 72, wherein the spatially labeled polynucleotide or a portion thereof is released from the further extended capture probe for analysis. 74. The method of claim 73, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing. 75. The method of any one of claims 64-74, wherein the method comprises imaging the spatial array using fluorescence microscopy. 76. The method of any one of claims 64-75, wherein the method comprises amplifying the released target RNA, the extended capture probe, the further extended capture probe, or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released target RNA, the extended capture probe, the further extended capture probe, or the spatially labeled polynucleotide, or an amplification product thereof. 77. The method of any one of claims 64-76, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array. 78. A method for analyzing a biological sample, the method comprising:^ny-2785575 202412020440 a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) hybridizing a probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; d) extending the hybridized probe to form an extended probe, wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) releasing the extended probe from the hydrogel matrix; f) hybridizing or ligating the released extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and g) determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. 79. The method of claim 78, wherein prior to a), the biological sample is a fresh frozen tissue sample or a decrosslinked tissue sample, or wherein prior to a), the biological sample is a fixed tissue sample, and between a) and b), the biological sample embedded in the hydrogel matrix is decrosslinked. 80. The method of claim 78 or claim 79, wherein the target RNA is 3’ tethered to the hydrogel matrix. 81. The method of claim 78 or claim 79, wherein the target RNA is 5’ tethered to the hydrogel matrix. 82. The method of any one of claims 78-81, wherein the probe hybridizes to a poly(A) sequence in the target RNA tethered to the hydrogel matrix. 83. The method of any one of claims 78-82, wherein the probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence. 84. The method of any one of claims 78-83, wherein the homopolymer sequence is a poly(C) sequence.^ny-2785575 202412020440 85. The method of any one of claims 78-84, wherein the capture domain in the capture probe comprises a template switch oligonucleotide sequence and a homopolymer sequence complementary to the homopolymer sequence at the 3’ end of the extended probe. 86. The method of claim 85, comprising extending the extended probe using the capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a complementary sequence of the released target RNA and a complementary sequence of the spatial barcode. 87. The method of claim 86, wherein the spatially labeled polynucleotide or a portion thereof is released from the capture probe for analysis. 88. The method of claim 87, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing. 89. The method of any one of claims 78-88, wherein the method comprises imaging the spatial array using fluorescence microscopy. 90. The method of any one of claims 78-89, wherein the method comprises amplifying the released target RNA, the extended probe, the capture probe, or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released target RNA, the extended probe, the capture probe, or the spatially labeled polynucleotide, or an amplification product thereof. 91. The method of any one of claims 78-90, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array. 92. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) hybridizing a first probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; d) extending the hybridized probe to form an extended probe, wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe;^ny-2785575 202412020440 e) hybridizing a second probe to the extended probe in the hydrogel matrix and further extending the extended probe using the second probe as a template to form a further extended probe, wherein the extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe; f) releasing the further extended probe comprising the double stranded region from the hydrogel matrix; g) hybridizing or ligating the released further extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and h) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the further extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. 93. The method of claim 92, wherein prior to a), the biological sample is a fresh frozen tissue sample or a decrosslinked tissue sample, or wherein prior to a), the biological sample is a fixed tissue sample, and between a) and b), the biological sample embedded in the hydrogel matrix is decrosslinked. 94. The method of claim 92 or claim 93, wherein the target RNA is 3’ tethered to the hydrogel matrix. 95. The method of claim 92 or claim 93, wherein the target RNA is 5’ tethered to the hydrogel matrix. 96. The method of any one of claims 92-95, wherein the first probe hybridizes to a poly(A) sequence in the target RNA tethered to the hydrogel matrix. 97. The method of any one of claims 92-96, wherein the first probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence. 98. The method of any one of claims 92-97, wherein the second probe comprises a 5’ poly(U) sequence, a template switch oligonucleotide sequence, and a homopolymer sequence complementary to the homopolymer sequence at the 3’ end of the extended probe.^ny-2785575 202412020440 99. The method of any one of claims 92-98, wherein the further extended probe comprises a 3’ poly(A) sequence. 100. The method of any one of claims 92-99, wherein the releasing in f) comprises releasing the tethered RNA from the hydrogel matrix. 101. The method of any one of claims 92-100, wherein the releasing in f) comprises releasing the further extended probe from the target RNA. 102. The method of any one of claims 92-101, wherein the releasing in f) comprises contacting the hydrogel matrix with an RNase H. 103. The method of any one of claims 92-102, wherein the capture domain in the capture probe comprises a 3’ poly(dT) sequence. 104. The method of any one of claims 92-103, wherein between g) and h), the method comprises generating a spatially labeled polynucleotide comprising: i) from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released further extended probe, or ii) from 5’ to 3’: a sequence of the released further extended probe and a complementary sequence of the spatial barcode. 105. The method of claim 104, wherein the spatially labeled polynucleotide or a portion thereof is released from the spatial array for analysis. 106. The method of claim 105, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing. 107. The method of any one of claims 92-106, wherein the method comprises imaging the spatial array using fluorescence microscopy. 108. The method of any one of claims 92-107, wherein the method comprises amplifying the released further extended probe or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released further extended probe or the spatially labeled polynucleotide or an amplification product thereof. 109. The method of any one of claims 92-108, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array. 162^ny-2785575 202412020440 110. The method of any one of claims 41-109, wherein the biological sample is contacted with a plurality of transposomes, wherein a transposome of the plurality of transposomes comprises (i) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, (ii) a linker, and (iii) a reactive group, thereby tagmenting an open chromatin in the biological sample with the plurality of transposomes, and wherein the tagmented open chromatin is tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin, wherein after the biological sample is removed from the hydrogel matrix, the tagmented open chromatin is released from the hydrogel matrix, the released tagmented open chromatin is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof is determined. 111. The method of any one of claims 41-110, wherein the biological sample is contacted with an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, and with a transposome-binding moiety complex that binds to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody-binding moiety, (iii) a transposon end sequence comprising a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, and (iv) a reactive group, thereby tagmenting a genomic DNA in the biological sample with the transposome-binding moiety complex, wherein the tagmented genomic DNA is tethered to the hydrogel matrix via the reactive group of the transposome-binding moiety complex, wherein after the biological sample is removed from the hydrogel matrix, the tagmented genomic DNA is released from the hydrogel matrix, the released tagmented genomic DNA is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.^ny-2785575 202412020440 112. The method of any one of claims 41-111, wherein the biological sample is contacted with an antibody-oligonucleotide conjugate, wherein the antibody-oligonucleotide conjugate comprises (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group, wherein the antibody-oligonucleotide conjugate is tethered to the hydrogel matrix via the reactive group of the antibody-oligonucleotide conjugate, wherein after the biological sample is removed from the hydrogel matrix, the antibody- oligonucleotide conjugate or a portion thereof is released from the hydrogel matrix, the capture sequence of the released antibody-oligonucleotide conjugate or portion thereof is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the barcode sequence associated with the antibody or a complement thereof and (ii) the spatial barcode or a complement thereof is determined. 113. The method of any one of claims 110-112, wherein two or more or all of the released ligation product in claim 41, the released target RNA in claim 64, the released extended probe in claim 78, the released further extended probe in claim 91, the released tagmented open chromatin in claim 109, the released tagmented genomic DNA in claim 110, the released antibody- oligonucleotide conjugate or portion thereof in claim 111 are hybridized or ligated to capture probes comprising capture domains of different sequences on the spatial array. 114. A method for analyzing a biological sample, comprising: a) contacting the biological sample with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (ii) a linker, and (iii) a reactive group; b) tagmenting an open chromatin in the biological sample with the plurality of transposomes; c) embedding the biological sample in a hydrogel matrix, wherein the tagmented open chromatin is tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin;^ny-2785575 202412020440 d) removing the biological sample from the hydrogel matrix; e) releasing the tagmented open chromatin from the hydrogel matrix, wherein the released tagmented open chromatin is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and f) determining a profile of the open chromatin in the biological sample by determining the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof. 115. Th method of claim 114, wherein the linker comprises an antibody-binding moiety, optionally wherein the antibody-binding moiety is protein A, protein G, or a fusion protein thereof; and/or wherein the capture sequence is in a first transposon end sequence, and the transposome or the transposome-binding moiety complex comprise a second transposon end sequence comprising a functional sequence, optionally wherein the functional sequence comprises a primer binding sequence or a complement thereof. 116. A method for analyzing a biological sample, comprising: a) contacting the biological sample with: an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, a transposome-binding moiety complex that binds to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody-binding moiety, (iii) a transposon end sequence comprising a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, and (iv) a reactive group; b) tagmenting a genomic DNA in the biological sample with the transposome- binding moiety complex; c) embedding the biological sample in a hydrogel matrix, wherein the tagmented genomic DNA is tethered to the hydrogel matrix via the reactive group of the transposome- binding moiety complex; d) removing the biological sample from the hydrogel matrix;^ny-2785575 202412020440 e) releasing the tagmented genomic DNA from the hydrogel matrix, wherein the released tagmented genomic DNA is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and f) determining the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof. 117. The method of claim 116, wherein the chromatin protein or chromatin-associated protein is a histone or a non-histone DNA binding protein, optionally wherein the non-histone DNA binding protein comprises a transcription factor or a transcription co-factor. 118. The method of claim 116 or claim 117, wherein the antibody binds to one or more epigenetic modifications, optionally wherein the antibody binds to one or more histone modifications. 119. The method of any one of claims 116-118, wherein the antibody directly binds to the chromatin protein or chromatin-associated protein, and the antibody-binding moiety in the transposome-binding moiety complex directly binds to the antibody. 120. The method of any one of claims 116-118, wherein the antibody is a primary antibody that directly binds to the chromatin protein or chromatin-associated protein, the biological sample is contacted with a secondary antibody that directly binds to the primary antibody, and the antibody-binding moiety in the transposome-binding moiety complex directly binds to the secondary antibody. 121. A method for analyzing a biological sample, comprising: a) contacting the biological sample with an antibody-oligonucleotide conjugate comprising (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group; b) embedding the biological sample in a hydrogel matrix, wherein the antibody- oligonucleotide conjugate is tethered to the hydrogel matrix via the reactive group of the antibody-oligonucleotide conjugate; c) removing the biological sample from the hydrogel matrix; d) releasing the antibody-oligonucleotide conjugate or a portion thereof from the hydrogel matrix, wherein capture sequence of the released antibody-oligonucleotide conjugate or 166^ny-2785575 202412020440 portion thereof is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and e) determining the sequence of (i) the barcode sequence associated with the antibody or a complement thereof and (ii) the spatial barcode or a complement thereof. 122. The method of any one of claims 110-121, wherein the reactive group comprises a 5’ acrylic phosphoramidite. 123. The method of any one of claims 110-122, wherein the transposome, the transposome- binding moiety complex, and/or the antibody-oligonucleotide conjugate comprises a cleavable linker connected to the reactive group. 124. The method of any one of claims 110-123, wherein the transposome, the transposome- binding moiety complex, and/or the antibody-oligonucleotide conjugate comprises a functional sequence. 125. The method of claim 124, wherein the functional sequence comprises a primer binding sequence or a complement thereof.^ny-2785575