CN113355750A - rRNA silent RNA library construction method and kit - Google Patents

Abstract

The invention provides a method for preparing rRNA silenced RNA libraries and reagents or kits used in the method.

Description

rRNA silent RNA library construction method and kit

Technical Field

The invention belongs to the technical field of biology, and relates to a method for preparing an rRNA silent RNA library and a reagent or a kit used by the method.

Background

The high throughput sequencing technology is also called second generation sequencing technology, can be abbreviated as NGS, and refers to a technology for performing sequencing on hundreds of thousands to millions of DNA molecules at a time, and the length of a determined sequence is generally short.

One major problem with transcriptome sequencing is the presence of interfering RNA molecules. For example, ribosomal RNA (rRNA) is the most abundant molecule in total RNA, with typically more than 90% of the total RNA being rRNA. However, ribosomal RNA provides little information about transcription. In applications such as RNA sequencing (RNA-seq), it is of great interest to maximize the amount of information received from the sequencing run. If the library construction involves abundant rRNA, most of the sequencing capacity will be used to sequence these ubiquitous molecules, thereby reducing the capacity available to study the remaining transcriptome. Thus, valuable sequencing resources are wasted. Furthermore, the presence of ribosomal RNA may result in a low signal-to-noise ratio, making it difficult to detect the RNA species of interest. Thus, removal of rRNA and/or other unwanted RNA improves the value of downstream sequencing. In order to provide sequencing libraries that do not contain rRNA or other undesirable RNA species, several methods have been developed in the prior art.

In recent years, researchers have developed various methods to remove non-target RNAs such as rRNA. The methods use the different aspects of RNA to distinguish and design removal means, such as size, sequence characteristics, 5' phosphate group, secondary structure, abundance and the like, which can be used as the basis of the removal method; also, there are several commercial kits available for removing non-target RNA.

First, it is noted that, in eukaryotes, since mRNA has the structure of poly (A) tail, it is easy to enrich and purify with poly (T) to remove other RNA, or synthesize cDNA directly with poly (T) primer; the mRNA of prokaryotes does not have the structure, so that non-target RNA in total RNA of prokaryotes is removed, and the technical difficulty is higher. Therefore, the rRNA removal methods described in detail below are generally applicable to both prokaryotes and eukaryotes.

PolyA RNA can be isolated by conventional methods, for example using magnetic beads functionalized with poly (T) oligonucleotides to capture the PolyA RNA accordingly. The advantage of preparing a sequencing library from polyA RNA is that RNA that does not carry polyA tails, such as rRNA, is not recovered from total RNA and, accordingly, is not carried over into the sequencing reaction. Thus, most of the sequences obtained from sequencing libraries generated using polyA RNA correspond to protein-encoding mrnas that do carry polyA tails. However, preparing sequencing libraries using purified polyA RNA also has disadvantages. Several types of RNA do not carry a polyA tail and are therefore lost in polyA enrichment, but are still of interest for transcriptome sequencing. polyA enrichment results in the loss of non-polyadenylated mRNA sequences that are an important component of the transcriptome. Some eukaryotic mrnas, such as those encoding histones, also do not carry polyA tails, others carry polyA tails that are too short to be efficiently captured by oligo dT. Furthermore, this method cannot be used for prokaryotic mrnas because they are not polyadenylated. Yet another disadvantage is that polyA enrichment requires high quality intact total RNA as input material. PolyA enrichment is not feasible for degraded RNA samples because only fragments carrying PolyA tails can be captured.

Another rRNA enzymatic technique utilizes rnase H to specifically degrade DNA: the ability of RNA to hybridize to RNA in a molecule without degrading RNA single strands (Hausen P, Stein H, Ribonuclase H. an enzyme digestion the RNAM oil of DNA-RNA hybrids. Eur J Biochem/FEBS 1970; 14 (2): 278-83). In this method, total RNA is first hybridized or reverse transcribed using a series of specific primer mixtures for rRNA, followed by the addition of rnase H to degrade DNA: the RNA hybridizes to rRNA in the double strand, followed by addition of DNase I to degrade the remaining DNA. JohnMorlan uses this principle to design short antisense DNA probes (50-80bp) directed against human/rat/mouse 5S, 18S, 28S rRNA. By hybridizing these probes with human RNA and treating with RNase H and DNase I, 98% of the rRNA can be removed. The disadvantages of the dnase I digestion technology based on rnase H are quite significant: first, since the primers used are designed for the target RNA, the probability of these primers not hybridizing to non-target RNA is extremely low, so that there is a high possibility that a part of non-target RNA is present in the final target RNA; secondly, the target RNA is obtained after a series of treatments of RNase H, DNase I and the like, the RNA is extremely easy to be polluted and degraded, and the more the target RNA removing steps are, the higher the possibility of degradation is.

Subtractive hybridization using specific probes subtractive hybridization uses the antisense sequence of rRNA as a specific probe so that after rRNA has been hybridized to probes bound to microspheres or magnetic beads, the hybridized molecules can be removed from solution. This method is currently the most widely used technique, such as the MICROB Express kit from Ambion, using two successive hybridizations to capture rRNA on magnetic beads. The kit has definite species limitation in use, and all archaea samples are not applicable; and limited by the type and number of probes used, the highly-degraded rRNA required by the kit for the integrity of RNA samples often loses hybridization sites and cannot be effectively removed. In addition, the later Ribo-Zero kit developed by Epicentre corporation used specific biotin-coupled probes, and rRNA was hybridized to these probes and then removed using streptavidin-coated affinity chromatography beads.

Disclosure of Invention

The invention aims to provide a simple and rapid rRNA silencing method and a kit for preparing an RNA sequencing library, compared with an RNase H digestion method, the steps are reduced, and the time is shortened from 3 hours to 15 minutes, so that the damage to RNA is reduced.

In one aspect, the invention provides a 5 'blocked rRNA-specific oligonucleotide having a 5' blocking moiety that blocks ligation (e.g., ligation of adaptors) at the 5 'end and a 3' rRNA-specific sequence (with or without additional sequences) that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to an rRNA target sequence. 5 'end closed means that the oligonucleotides of the invention are not capable of ligating adaptors as described herein at the 5' end, such that dsDNA derived from rRNA cannot be efficiently amplified using adaptor-specific primers, due to the absence of ligated adaptors, and subsequently formed into a library, e.g., for sequencing.

In one embodiment, the 5 'end blocking moiety is a modification of the 5' -phosphate group or the 5 '-hydroxyl group of the 5' terminal nucleotide. In a further embodiment, the modification is esterification or amidation of the 5 '-phosphate group or esterification or etherification of the 5' -hydroxyl group. In a further embodiment, the modification is a 5 'position linker spacer sequence, e.g., 5' spacer 18, 5 'spacer 9, 5' C3-spacer, 5 'C6-spacer, 5' no base residue (d spacer, r spacer), 5 '-5' inverted nucleotide. In one embodiment, the base of the 5' terminal nucleotide is not any of thymine, adenine, cytosine, guanine and uracil. In one embodiment, one or both of the 2 'hydrogens of the deoxyribose sugar of the 5' terminal nucleotide is replaced with another atom or blocking moiety. In one embodiment, the oligonucleotide comprises a 5' terminal nucleotide having a pentose sugar in a conformation different from that of ribose or deoxyribose in RNA or DNA. For 5' blocking see WO2017/032808 or CN107849561A, which are incorporated herein by reference for all purposes.

In another embodiment, the 3' end rRNA specific sequence and/or the rRNA target sequence has a length, e.g., from 4nt to 100nt, from 6nt to 75nt, from 10nt to 60nt, from 20nt to 50nt, or from 30nt to 40nt, including any integer in the range, e.g., a length of 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, or 75 nt.

In another embodiment, the rRNA-specific sequence is specific for 28S, 26S, 25S, 18S, 5.8S, or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S, or 5S prokaryotic rRNA, and/or the rRNA target sequence is from 28S, 26S, 25S, 18S, 5.8S, or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S, or 5S prokaryotic rRNA.

In another embodiment, the rRNA target sequence is a conserved sequence or a consensus sequence of rRNA.

In another embodiment, the 3' terminal rRNA specific sequence comprises degenerate nucleotides.

In another embodiment, the base of the oligonucleotide is modified, e.g., 5-Methyl dC, Super T and/or 2, 6-Amino-dA, capable of stably annealing to rRNA to form a hybrid strand and extending. In a further embodiment, all of the C's in the oligonucleotide are modified. In a further embodiment, all of the T's in the oligonucleotide are modified. In a further embodiment, all a's in the oligonucleotide are modified.

In another embodiment, the rRNA is from a species of interest, including but not limited to a mammal, such as a primate or rodent, and further such as a human, cynomolgus monkey, mouse, rat, rabbit, dog, cat, cow, horse, sheep, pig.

In another embodiment, the rRNA is from a microorganism of interest, such as a bacterium or fungus, including but not limited to escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, saccharomyces cerevisiae, pichia pastoris.

In another embodiment, the oligonucleotide comprises any one selected from SEQ ID No.1 to SEQ ID No. 119.

In another aspect, the invention provides a collection of oligonucleotides comprising a plurality of oligonucleotides of the invention and specific for the same rRNA class, e.g., one of 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or 23S, 16S or 5S prokaryotic rRNA.

In one embodiment, the rRNA target sequences of the plurality of oligonucleotides overlap, are adjacent, are discontinuous, or any combination thereof, with each other on the rRNA. In one embodiment, the rRNA target sequences of the plurality of oligonucleotides are uniformly, near-uniformly (e.g., with a deviation from uniformity of less than about 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1%, calculated based on the length of the rRNA target sequence) or non-uniformly distributed on the rRNA. In one embodiment of rRNA target sequence overlap, the (average) length of the overlapping portion is at least about 5%, 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100% of the (average) length of the rRNA target sequence. In one embodiment of rRNA target sequence discontinuity, the (average) length of non-target rRNA sequences (i.e., sequences between two adjacent rRNA target sequences on an rRNA) is at least about 5%, 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 175%, 200% of the (average) length of the rRNA target sequences.

In another embodiment, the 3' end rRNA specific sequences and/or rRNA target sequences of the collection of oligonucleotides have, for example, an average length of about 4nt to 100nt, 6nt to 75nt, 10nt to 60nt, 20nt to 50nt, or 30nt to 40nt, including any number within the stated range, such as an average length of about 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, or 75 nt.

In another embodiment, the rRNA target sequences of the collection of oligonucleotides cover at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% of the full length of the rrnas. Coverage refers to the percentage of the pool of rRNA target sequences over the full length of the rRNA sequences.

In another embodiment, the collection of oligonucleotides comprises any one selected from the group consisting of SEQ ID No.1 to SEQ ID No.2, SEQ ID No.3 to SEQ ID No.5, SEQ ID No.6 to SEQ ID No.19, or SEQ ID No.20 to SEQ ID No. 119.

In another aspect, the invention provides an oligonucleotide mixture comprising at least two, three, four, five, six, seven, eight or more pools of oligonucleotides of the invention, the first pool of oligonucleotides being specific for a first type of rRNA, e.g., 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or one of 23S, 16S or 5S prokaryotic rRNA, the second pool of oligonucleotides being specific for a second type of rRNA, e.g., 28S, 26S, 25S, 18S, 5.8S or 5S eukaryotic cytoplasmic rRNA or 16S or 12S eukaryotic mitochondrial rRNA or one of 23S, 16S or 5S prokaryotic rRNA, the first type of rRNA being different from the second type of rRNA, and so on.

In one embodiment, the oligonucleotide mixture comprises a plurality of sets of oligonucleotides specific for each class of all eukaryotic rRNA and/or all prokaryotic rRNA, i.e., 28S, 26S, 25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNA and/or each class of 16S and 12S eukaryotic mitochondrial rRNA and/or each class of 23S, 16S and 5S prokaryotic rRNA.

In another embodiment, the oligonucleotide mixture comprises any two or more groups selected from the group consisting of SEQ ID No.1 to SEQ ID No.2, SEQ ID No.3 to SEQ ID No.5, SEQ ID No.6 to SEQ ID No.19, and/or SEQ ID No.20 to SEQ ID No. 119.

In one embodiment of any of the above aspects, the oligonucleotide, collection of oligonucleotides or mixture of oligonucleotides is used as a primer.

In another aspect, the invention provides a method of preparing an rRNA-silenced RNA library (e.g., for sequencing), comprising one or more of the following steps:

optionally, fragmenting the RNA sample;

reverse transcription is carried out by using the rRNA specific oligonucleotide or the oligonucleotide set or the oligonucleotide mixture with the 5' end blocked as a rRNA primer, a random oligonucleotide (such as a random hexamer) as a primer of other RNA and a reverse transcriptase to obtain a first strand of cDNA;

synthesizing a second strand of DNA using random oligonucleotides (e.g., random hexamers) and a DNA polymerase;

connecting an adaptor head;

optionally, recovering and purifying the ligation product; and

optionally, the ligation product is amplified.

In one embodiment, the adaptor is a sequencing adaptor. In one embodiment, the sequencing adaptor is a Y-type sequencing adaptor. In one embodiment, the sequencing adaptor is a sequencing adaptor of the illumina platform, a sequencing adaptor of the Ion Torrent platform, or a sequencing adaptor of the huada MGI platform.

In one embodiment, the amount (in moles) of the 5' blocked rRNA-specific oligonucleotide or collection of oligonucleotides or mixture of oligonucleotides added is at least about 0.01x, 0.02x, 0.05x, 0.1x, 0.2x, 0.5x, 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 10x, 20x, 50x, 100x of the amount (measured or estimated) of rRNA or fragments thereof. In one embodiment, the amount (by mass) of the 5' blocked rRNA-specific oligonucleotide or collection of oligonucleotides or mixture of oligonucleotides added is at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 33%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 1x, 2x, 3x, 5x, 10x, 20x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 500x, 1000x of the amount (measured or estimated) of rRNA or fragment thereof.

In another aspect, the invention provides a kit comprising a 5' blocked rRNA specific oligonucleotide or collection of oligonucleotides or mixture of oligonucleotides of the invention. In one embodiment, the kit further comprises other reagents required for carrying out the method of the invention. In another embodiment, the kit is used to perform the methods of the invention, e.g., for making rRNA-silenced RNA libraries. In a further embodiment, the RNA library is used for sequencing.

In another embodiment, the method or library constructed according to the invention is chain-specific. In another embodiment, the invention can distinguish between forward and reverse transcripts, for example by sequence of an adaptor. That is, the present invention can construct a forward transcript library or a reverse transcript library.

The implementation scheme of the invention comprises the following steps:

(1) RNA fragmentation

Total RNA is fragmented into fragments of appropriate length or range of lengths, typically non-sequence or site-specific. For example, by fragmenting the total RNA using divalent cations under high temperature conditions, the length or length of the fragments can be in the range of about 50-1000nt, such as 100-500nt, e.g., 100-200, 200-300, 300-400, 400-500, 100-300, 200-400, or 300-500 nt. The reaction temperature and time of fragmentation can be adjusted to obtain different lengths or ranges of lengths.

(2) Hybridization of primers

The fragmented rRNA fragments were bound to 5' -blocked rRNA-specific oligonucleotides. The blocked 5' end is not ligated to the adaptors described herein.

At the same time, random primers (e.g., random hexamers) free at the 5' end bind to fragments of other RNAs (e.g., mRNAs). The free 5' end is capable of attaching an adaptor as described herein.

(3) One-chain synthesis

And (3) carrying out extension reaction on the hybridized product in the step (2) by using reverse transcriptase to obtain a DNA-RNA hybrid. Optionally, after synthesis of the first strand, purification may be performed, and/or the RNA strand in the DNA-RNA hybrid may be digested.

(4) Double chain synthesis

Two-strand DNA synthesis is carried out using a DNA polymerase (e.g.a high fidelity DNA polymerase, i.e.a DNA polymerase having proofreading activity, i.e.3 ' -5 ' exo activity, such as Pfu and T4 DNA polymerase) and random primers which are free at the 5 ' end (e.g.random hexamers). Optionally, polynucleotide kinases are also included. Optionally, enzymes with terminal transferase activity (e.g., Taq) are also included. Preferably, the resulting terminus comprises an adenosine for ligation to an adaptor with thymidine. Optionally, the second strand synthesis may employ U and digest away the U-containing second strand at the appropriate time. Optionally, purification may be performed after the second strand is synthesized.

(5) Adapter connection

According to the requirement (such as a solid phase and a primer used for sequencing), a corresponding adaptor (such as a Y-type sequencing adaptor) is selected to be connected with the double-stranded DNA obtained in the step (4). In this case, the 5 '-end of the double strand derived from rRNA as a template by using the rRNA-specific oligonucleotide having the 5' -end blocked cannot be ligated to the adaptor, and the 5 '-end of the double strand derived from another RNA as a template by using the random primer having the 5' -end released can be ligated to the adaptor. Optionally, the ligation product is purified, for example using magnetic beads. Optionally, the ligation product is amplified, e.g., by PCR, without purification or after purification.

The above steps can be combined arbitrarily according to the situation. The product thus obtained can be used for sequencing, for example second generation sequencing. For another example, only the first strand is sequenced, particularly the first strand derived from an RNA other than rRNA.

In another aspect, the invention provides a kit for preparing an rRNA-silenced RNA library, comprising one or more of the following reagents: a disruption buffer, rRNA specific primers (e.g., 5 'blocked) and random primers (e.g., 5' free), reverse transcriptase, reverse transcription buffer solution, duplex synthetase mix, duplex synthesis buffer, RNA purification magnetic beads, DNA purification magnetic beads, adaptors (e.g., sequencing adaptors), ligase, ligation buffer, high fidelity DNA polymerase, amplification buffer, amplification primers, and nuclease-free water.

The above reagents may be combined as appropriate. For example, reverse transcriptase, reverse transcription buffer solution can be mixed as a mixture. For another example, the amplification buffer and the amplification primers may be mixed together as a single mixture. Additionally or alternatively, RNA purification magnetic beads, DNA purification magnetic beads, amplification primers, nuclease-free water may or may not be present in the kit, i.e. provided externally.

The kit can eliminate rRNA from total RNA, retain mRNA and other non-coding RNA, and can be used for analysis of non-coding RNA such as LncRNA.

Degraded RNA samples can also be subjected to library construction using the kit.

The present invention is applicable to total RNA samples with an initial amount of 1-1000 ng.

Brief Description of Drawings

FIG. 1 shows the distribution pattern of rRNA target sequences of the present invention. Panel A shows a schematic representation of the target rRNA. Panel B shows schematic representations of the overlap of rRNA target sequences with each other. Panel C shows schematic representations of rRNA target sequences adjacent to each other. Panel D shows a schematic representation of the interruption of rRNA target sequences to each other.

FIG. 2 shows a flow chart of the method of the present invention.

FIG. 3 shows the results of Agilent 2100 Bioanalyzer analysis of libraries prepared according to the invention and controls.

Detailed Description

rRNA removal Using Ribo-zero rRNA removal kit of Illumina and the like

Stranded Total RNA Sample Preparation kits were pooled as controls.

In this example, 1000ng of total RNA extracted from conventionally cultured HEK293 cells was used as a starting sample for rRNA silencing and construction of a transcriptome library for sequencing, as follows:

1. RNA fragmentation and primer hybridization

1000ng of total RNA was treated with RNase-free ddH₂O was diluted to 7. mu.l.

Preparing the following mixed solution in an RNase-free centrifuge tube:

TABLE 1

^aNanjing Novozam Biotech Inc. (vazyme, # N402) contains random primers.

^bSee the sequence table. The concentration of each primer was 50. mu.M. All primers are modified with a C3 spacer at the 5' end. All A, T, C bases were modified: c, 5-Methyl dC; t, Super T; a, 2, 6-Amino-dA.

The following reaction was carried out:

TABLE 2

Temperature of	Time
		85℃	6min
75℃	2min
		70℃	2min
65℃	2min
		60℃	2min
55℃	2min
		37℃	2min
25℃	2min
		4℃	Holding

2. Reverse transcription

Preparing a reverse transcription reaction system:

TABLE 3

^aVAHTS Universal V6 RNA-seq library Prep Kit for Illu, Biotech, Inc. of Nanjing NovowedAnd (5) a mina kit.

Reverse transcription reaction conditions:

TABLE 4

Temperature of	Time
		25℃	10min
42℃	15min
		70℃	15min
4℃	Holding

The second strand synthesis reaction was immediately performed.

3. Double chain synthesis

Preparing a mixed solution:

TABLE 5

^aKit for Illumina, VaHTS Universal V6 RNA-seq library Prep Kit, Nyjinomo Scienda Biotech, Inc.

The following reaction was carried out:

TABLE 6

Temperature of	Time
		16℃	30min
65℃	15min
		4℃	Holding

4. Adapter connection

Taking the Adapter out of the reaction kettle at the temperature of-20 ℃, thawing, reversing and uniformly mixing to prepare a ligation reaction solution:

TABLE 7

^aKit for Illumina, VaHTS Universal V6 RNA-seq library Prep Kit, Nyjinomo Scienda Biotech, Inc.

^bTake the adaptor of the Illumina platform as an example.

The following reaction was carried out:

TABLE 8

Temperature of	Time
		20℃	15min
4℃	Holding

5. Ligation product purification and fragment size sorting

Ligation products were purified using VAHTS DNA Clean Beads (Biotech, Inc., Nuo Wei Zan, Nanjing) according to the instructions.

A library of 550bp 450-.

6. Library amplification

And preparing a PCR reaction solution.

TABLE 9

^aKit for Illumina, VaHTS Universal V6 RNA-seq library Prep Kit, Nyjinomo Scienda Biotech, Inc.

The sample was placed in a PCR apparatus and the following reaction was carried out

Watch 10

7. Amplification product purification

Ligation products were purified using VAHTS DNA Clean Beads according to the instructions.

Qubit assay library concentration

The resulting library was subjected to concentration determination using Qubit and library yield was calculated. The results are shown in the following table.

TABLE 11

Sample preparation method	Library Generation (ng/. mu.l)
		The invention	38.6
Control (Ribo-zero)	37.9

9. Evaluation of library quality with Agilent 2100 Bioanalyzer

Mu.l of the purified PCR product was taken and analyzed with Agilent DNA 1000 kit (Agilent, Cat. No. 5067-1504). The results are shown in FIG. 3 (A: present invention, B: Ribo-zero).

10. Sequencing

And (3) carrying out machine sequencing on an Illumina platform, and carrying out data analysis to obtain the data in the following table.

Table 12: specific sequencing data Condition

As can be seen from the data in Table 12, from the information of Reads number, GC content, Q20, Q30 number and the like, the data of the two libraries are basically consistent, and the sequencing indexes of the libraries are proved to be normal; aligning the sequencing data to a human reference genome, both libraries having a high percentage of alignment (MappingRate); the library quality is proved to be good; from the information of DuplicationRate, detection gene factors and the like, the library obtained by the invention has lower redundancy rate and higher gene detection number; as can be seen from the rRNA ratio, the method has unsophisticated rRNA removal efficiency, reduces data waste and can obtain more data information.

This example achieved a reduction in the proportion of rRNA from more than 90% in total RNA samples to 1.01% in the sequencing results using the primer mix listed in the appendix. In contrast, the control method decreased to 5.32%. By optimizing the library building conditions, such as increasing the number of primers, improving the coverage rate of the target sequence, optimizing the selection of the target sequence, and improving the addition amount of the primers relative to the sample, the rRNA proportion in the sequencing result can be further reduced, even completely eliminated.

To sum up: the invention provides a novel rRNA removal RNA library construction scheme, the operation of which is not much different from the standard library construction process, no additional enzyme reaction is introduced, a high-quality library can be obtained, and the rRNA removal efficiency is high.

Sequence listing

<110> Nanjing Novozan Biotechnology GmbH

<120> rRNA silent RNA library construction method and kit

<130> 2021.01.06

<160> 119

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<223> 28s primer 27-77

<400> 20

tcctggttag tttcttttcc tccgctgact aatatgctta aattcagcgg 50

<210> 21

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 90-130

<400> 21

ggcggggatt cggcgctggg ctcttccctg ttcactcgcc 40

<210> 22

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 133-

<400> 22

ggggagcggg tcttccgtac gccacatgtc ccgcgccccg 40

<210> 23

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 177-

<400> 23

tcacaccgtc cacgggctgg gcctcgatca gaaggacttg ggccccccac gagcgg 56

<210> 24

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 245-

<400> 24

ctgcattccc aagcaacccg actccgggaa gacccgggcg cgcgccgggg g 51

<210> 25

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 309-359

<400> 25

cttgttgact atcggtctcg tgccggtatt tagccttaga tggagtttac 50

<210> 26

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 378-428

<400> 26

tacctcttaa cggtttcacg ccctcttgaa ctctctcttc aaagttcttt 50

<210> 27

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 443-

<400> 27

ccggacccgc cgccgggttg aatcctccgg gcggactgcg 40

<210> 28

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 514-

<400> 28

gggaagggag ggcgggtgga ggggtcggga ggaacggggg 40

<210> 29

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 562-

<400> 29

cacccgccgg agcccgcccc ctccggggag gaggaggagg 40

<210> 30

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 606-

<400> 30

gacggtcccc cgccgacccc acccccggcc ccgcccgccc acc 43

<210> 31

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 680-726

<400> 31

cttcccagcc gtcccggagc cggtcgcggc gcaccgcctg gaaatg 46

<210> 32

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 734-

<400> 32

gaggaggagg acggacggac ggacggggcc ccccgagcca ccttccc 47

<210> 33

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 765-805

<400> 33

cggggccggg gggcggagac gggggaggag gaggacggac 40

<210> 34

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 826-878

<400> 34

cgccgccccc gccgccgccg ccaccgccgc cgccgccgcc gccccgaccc gc 52

<210> 35

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 898-955

<400> 35

gtctcgctcc ctcggccccg ggattcggcg agtgctgctg ccgggggggc tgtaaca 57

<210> 36

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 973-

<400> 36

cccctcgcgg ggattcccgc gggggtgggc gccgggaggg 40

<210> 37

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1012-

<400> 37

cccccacgag gagacgccgg cgccgcgccg ggggagaccc 40

<210> 38

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1059-

<400> 38

gaggaggggt gggagagcgg tcgcgccgtg ggaggggtgg 40

<210> 39

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1091-1131

<400> 39

cacccccccc gtcgccgggg cgggggcgcg gggaggaggg 40

<210> 40

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1157-1197

<400> 40

cccgacggcg cgacccgccc ggggcgcact ggggacagtc 40

<210> 41

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1202-

<400> 41

ctcttcgggg gacgcgcgcg tggccccgag agaacctccc 40

<210> 42

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1235-1288

<400> 42

gtagccgacg tcgccgccga ccccgtgcgc tcgctccgcc gtccccctct tcg 53

<210> 43

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1299-1339

<400> 43

ctcgcgcacg tgttagactc cttggtccgt gtttcaagac 40

<210> 44

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1344-1384

<400> 44

ccttcacctt cattgcgcca cggcggcttt cgtgcgagcc 40

<210> 45

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1418-

<400> 45

cgagacgggc cggtggtgcc cctcggcgga ctggagaggc 40

<210> 46

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1481-1522

<400> 46

catagttcac catctttcgg gtcctaacac gtgcgctcgt g 41

<210> 47

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1528-1585

<400> 47

atttgcacgt caggaccgct acggacctcc accagagttt cctctggctt cgccctg 57

<210> 48

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1610-

<400> 48

gaaacttcgg agggaaccag ctactagatg gttcgattag tctttcg 47

<210> 49

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1684-1738

<400> 49

gacctctaat cattcgcttt accggataaa actgcgtggc gggggtgcgt cggg 54

<210> 50

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1756-

<400> 50

cagcgagccg ggcttcttac ccatttaaag tttgagaata ggttg 45

<210> 51

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1801-1858

<400> 51

agttctgctt accaaaagtg gcccactagg cactcgcatt ccaccccggc tccacgc 57

<210> 52

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1886-

<400> 52

tctatatcaa ccaacacctt ttctggggtc tgatgagcgt cggcatcggg cgccttaa 58

<210> 53

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 1954-1998

<400> 53

gagttgttac acactcctta gcggattccg acttccatgg ccac 44

<210> 54

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2020-

<400> 54

ggccgggtat gggcccgacg ctccagcgcc atccattttc 40

<210> 55

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2071-

<400> 55

tccgacgcac accacacgcg cgcgcgcgcc gccgcccccg ccgctcccgt ccactctcga 60

<210> 56

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2144-

<400> 56

ggggggggcg ggggaaggac cccacacccc cgccgccgcc 40

<210> 57

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2182-2222

<400> 57

gagcggggcg tgggcgggag gaggggagga ggcgtggggg 40

<210> 58

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2222-

<400> 58

ctcctactcg tcgcggcgta gctccgcggg gctccggggg cggg 44

<210> 59

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2291-2349

<400> 59

tttgctacta ccaccaagat ctgcacctgc ggcggcctcc acccgggccc gcgcccta 58

<210> 60

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2359-2413

<400> 60

atgttcaact gctgttcaca tggaaccctt ctccacttcg gccttcaaag ttct 54

<210> 61

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2422-2462

<400> 61

cccgtccctt cggaacggcg ctcgcccatc tctcaggacc 40

<210> 62

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2456-2511

<400> 62

atctgaaccc gactcccttt cgatcggccg agggcaacgg aggccatcgc ccgtc 55

<210> 63

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2496-

<400> 63

catctccgcc actccggatt cggggatctg aacccgactc 40

<210> 64

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2543-

<400> 64

cttctccggg atcggtcgcg ttaccgcact ggacgcctcg 40

<210> 65

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2615-

<400> 65

ctctctcggg gcgaacccat tccagggcgc cctgcccttc acaaa 45

<210> 66

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2653-2693

<400> 66

ccggaaccgc gacgctttcc aaggcacggg cccctctctc 40

<210> 67

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2722-2772

<400> 67

gatatgggta cggcccggcg cgagatttac accctctccc ccggattttc 50

<210> 68

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2762-2821

<400> 68

tacattgttc caacatgcca gaggctgttc accttggaga cctgctgcgg atatgggta 59

<210> 69

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2836-2877

<400> 69

cttagagcca atccttatcc cgaagttacg gatccggctt g 41

<210> 70

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2866-

<400> 70

cttcgcgccc cagcccgacc gacccagccc ttagagccaa 40

<210> 71

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2902-

<400> 71

cgcgcctcgt ccagccgcgg cgcgcgccca gccccgcttc 40

<210> 72

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 2932-

<400> 72

ggagggccgc gaggggggtg ccccgggcgt ggggggggcg cgcgcctcgt 50

<210> 73

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3002-3042

<400> 73

agagagagag agagggcgcg gggtggggag ggagcgagcg 40

<210> 74

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3050-3090

<400> 74

cccacgcggc gctcccccgg ggagggggga ggacggggag 40

<210> 75

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3084-

<400> 75

cctgccgccc cgacccttct ccccccgccg cgcccccacg 40

<210> 76

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3119-3172

<400> 76

tcgcggggac ctgcccccgc cggccgcccc ggcggccgcc gcgcggcccc tgc 53

<210> 77

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3149-

<400> 77

ggtccccggg gcccccctcg cggggacctg cccccgccgg 40

<210> 78

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3190-

<400> 78

tccacgggaa gggcccggct cgcgtccaga gtccgcgccg ccgccggccc ccc 53

<210> 79

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3230-3270

<400> 79

ccgcgacgcc cgccgcagct ggggcgatcc acgggaaggg 40

<210> 80

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3298-3351

<400> 80

ccccagcgga cgcgcgcgcg accgagacgt ggggtggggg tggggggcgc gcc 53

<210> 81

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3329-3384

<400> 81

gcccgccgac cgccgccgcc cgaccgctcc cgcccccagc ggacgcgcgc gcgac 55

<210> 82

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3360-3405

<400> 82

gacgaaccgc cccgccccgc cgcccgccga ccgccgccgc ccgac 45

<210> 83

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3392-3432

<400> 83

gacggggccg ggggggtagg gcggggggac gaaccgcccc 40

<210> 84

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3436-3476

<400> 84

gccgccgccg ccgcgcgccg aggaggaggg gggaacgggg 40

<210> 85

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3482-3522

<400> 85

cggacccggc gggggggacc ggcccgcggc ccctccgccg 40

<210> 86

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3521-3577

<400> 86

tcggctgcta ggcgccggcc gaggcgaggc gcgcgcggaa ccgcggcccc gggggc 56

<210> 87

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3560-3616

<400> 87

ttaaacagtc ggattcccct ggtccgcacc agttctaagt cggctgctag gcgccg 56

<210> 88

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3599-3640

<400> 88

ggccttcgcg atgctttgtt ttaattaaac agtcggattc c 41

<210> 89

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3640-

<400> 89

ttcttcactt tgacattcag agcactgggc agaaatcaca tcgcgtcaac acccgccgcg 60

<210> 90

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3672-3722

<400> 90

gtttacccgc gcttcattga atttcttcac tttgacattc agagcactgg 50

<210> 91

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3717-3767

<400> 91

gacgaggcat ttggctacct taagagagtc atagttactc ccgccgttta 50

<210> 92

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3755-3805

<400> 92

aatctcgttc atccattcat gcgcgtcact aattagatga cgaggcattt 50

<210> 93

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3785-3835

<400> 93

tttcgctgga tagtaggtag ggacagtggg aatctcgttc atccattcat 50

<210> 94

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3854-3906

<400> 94

cagactagag tcaagctcaa cagggtcttc tttccccgct gattccgcca ag 52

<210> 95

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3891-

<400> 95

acttattcta cacctctcat gtctcttcac cgtgccagac tagagtcaag 50

<210> 96

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 3956-

<400> 96

cggaccccgc cccgggcccc tcgcggggac accggggggg 40

<210> 97

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4019-

<400> 97

gcctcaccgg gtcagtgaaa aaacgatcag agtagtggta t 41

<210> 98

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4068-

<400> 98

ggcgggcgct tggcgccaga agcgagagcc cctcgggctc 40

<210> 99

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4098-4151

<400> 99

ctccccacct ggcactgtcc ccggagcggg tcgcgcccgg ccgggcgggc gct 53

<210> 100

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4164-

<400> 100

ctcgccttag gacacctgcg ttaccgtttg acaggtgtac 40

<210> 101

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4210-

<400> 101

aaaatcaaga tcaagcgagc ttttgccctt ctgctccacg ggaggtttct gtcctc 56

<210> 102

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4272-4326

<400> 102

taaaacccaa aaggtcagaa ggatcgtgag gccccgcttt cacggtctgt attc 54

<210> 103

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4305-

<400> 103

ctgtggtaac ttttctgaca cctcctgctt aaaacccaaa aggtcagaag 50

<210> 104

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4339-

<400> 104

ctatgaacgc ttggccgcca caagccagtt atccctgtgg taacttttct 50

<210> 105

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4382-4438

<400> 105

ttcacaatga taggaagagc cgacatcgaa ggatcaaaaa gcgacgtcgc tatgaa 56

<210> 106

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4419-4468

<400> 106

gtgaacaatc caacgcttgg cgaattctgc ttcacaatga taggaagag 49

<210> 107

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4450-

<400> 107

gtctaaaccc agctcacgtt ccctattagt gggtgaacaa tccaacgctt 50

<210> 108

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4492-4542

<400> 108

caacacatca tcagtagggt aaaactaacc tgtctcacga cggtctaaac 50

<210> 109

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4525-4575

<400> 109

ttcctctcgt actgagcagg attaccatgg caacaacaca tcatcagtag 50

<210> 110

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4581-4622

<400> 110

cattggctcc tcagccaagc acatacacca aatgtctgaa c 41

<210> 111

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4626-4675

<400> 111

ggattctgac ttagaggcgt tcagtcataa tcccacagat ggtagcttc 49

<210> 112

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4659-4699

<400> 112

ctgccgtatc gttccgcctg ggcgggattc tgacttagag 40

<210> 113

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4706-

<400> 113

gacaggcggg ggaccggcta tccgaggcca accgaggctc 40

<210> 114

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4768-

<400> 114

ccggtcccgg cgcgcggcgg ggcacgcgcc ctcccgcggc ggggcgcgtg gag 53

<210> 115

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4815-

<400> 115

gtttcccagg acgaagggca ctccgcaccg gaccccggtc 40

<210> 116

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4847-

<400> 116

cgagggggcg gccgcctttc cggccgcgcc ccgtttccca 40

<210> 117

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4880-4934

<400> 117

gaatggttta gcgccaggtt ccccacgaac gtgcggtgcg tgacgggcga gggg 54

<210> 118

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4943-5003

<400> 118

ttcaatagat cgcagcgagg gagctgctct gctacgtacg aaaccccgac ccagaagcag 60

<210> 119

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> 28s primer 4988-

<400> 119

caaacccttg tgtcgagggc tgactttcaa tagatcgcag 40

Claims (10)

1. A5 'blocked rRNA specific oligonucleotide having a 5' blocking moiety that blocks ligation at the 5 'terminus and a 3' terminal rRNA specific sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to an rRNA target sequence,

optionally, the 5 ' end block module is a modification to the 5 ' -phosphate group or the 5 ' -hydroxyl group of the 5 ' terminal nucleotide, further optionally, the modification is esterification or amidation of the 5 ' -phosphate group or esterification or etherification of the 5 ' -hydroxyl group, or, the modification is a 5 ' position ligation spacer sequence,

optionally, the 3' end rRNA specific sequence and/or the rRNA target sequence has a length, e.g., from 4nt to 100nt, from 6nt to 75nt, from 10nt to 60nt, from 20nt to 50nt, or from 30nt to 40nt, including the length of any integer in the range, e.g., 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, or 75 nt.

2. The oligonucleotide of claim 1, wherein the rRNA specific sequence is specific for 28S, 26S, 25S, 18S, 16S, 12S, 5.8S or 5S eukaryotic rRNA or 23S, 16S or 5S prokaryotic rRNA, and/or the rRNA target sequence is from 28S, 26S, 25S, 18S, 16S, 12S, 5.8S or 5S eukaryotic rRNA or 23S, 16S or 5S prokaryotic rRNA.

3. The oligonucleotide of claim 1, wherein the rRNA target sequence is a conserved sequence or a consensus sequence of rRNA.

4. The oligonucleotide according to claim 1, wherein the 3' terminal rRNA specific sequence comprises degenerate nucleotides and/or modified bases.

5. A collection of oligonucleotides comprising a plurality of oligonucleotides according to any one of claims 1 to 4 and being specific for one of the same rRNA species, such as 28S, 26S, 25S, 18S, 16S, 12S, 5.8S or 5S eukaryotic rRNA or 23S, 16S or 5S prokaryotic rRNA,

optionally, the rRNA target sequences of the plurality of oligonucleotides overlap, are adjacent, are discontinuous, or any combination thereof, with each other on the rRNAs,

optionally, the rRNA target sequences of the plurality of oligonucleotides are distributed uniformly or non-uniformly over the rRNA,

optionally, the 3' end rRNA specific sequences and/or rRNA target sequences of the collection of oligonucleotides have, for example, an average length of about 4nt to 100nt, 6nt to 75nt, 10nt to 60nt, 20nt to 50nt, or 30nt to 40nt, including any number within the stated ranges, such as an average length of about 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, or 75nt,

optionally, the rRNA target sequences of the set of oligonucleotides cover at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% of the full length of the rrnas.

6. A mixture of oligonucleotides comprising at least two, three, four, five, six, seven, eight or more sets of oligonucleotides according to claim 5, a first set of oligonucleotides being specific for a first type of rRNA, such as one of 28S, 26S, 25S, 18S, 16S, 12S, 5.8S or 5S eukaryotic rRNA or 23S, 16S or 5S prokaryotic rRNA, a second set of oligonucleotides being specific for a second type of rRNA, such as one of 28S, 26S, 25S, 18S, 16S, 12S, 5.8S or 5S eukaryotic rRNA or 23S, 16S or 5S prokaryotic rRNA, the first type of rRNA being different from the second type of rRNA, and so on,

optionally, the oligonucleotide mixture comprises a plurality of oligonucleotide sets specific for all eukaryotic rrnas and/or all prokaryotic rrnas.

7. An oligonucleotide according to any one of claims 1 to 4 or a collection of oligonucleotides according to claim 5 or a mixture of oligonucleotides according to claim 6 for use as a primer.

8. A method of preparing an rRNA-silenced RNA sequencing library, comprising one or more of the following steps:

optionally, fragmenting the RNA sequencing sample;

performing reverse transcription using the collection of oligonucleotides according to claim 5 or the mixture of oligonucleotides according to claim 6, a random primer (e.g., a random hexamer), and a reverse transcriptase to obtain a first strand of cDNA;

synthesizing a second strand of DNA using random oligonucleotides (e.g., random hexamers) and a DNA polymerase;

ligating sequencing adaptors, optionally the sequencing adaptors are Y-type sequencing adaptors, optionally the sequencing adaptors are sequencing adaptors of an illumina platform, sequencing adaptors of an Ion Torrent platform or sequencing adaptors of a Huada Gongzhi MGI platform;

optionally, recovering and purifying the ligation product; and

optionally, the ligation product is amplified.

9. The method of claim 8, further comprising sequencing the ligation product.

10. A kit comprising a collection of oligonucleotides according to claim 5 or a mixture of oligonucleotides according to claim 6, optionally for performing a method according to claim 8.

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