Full-automatic RNA library preparation device applied to second-generation high-throughput sequencing(I) technical field
The invention belongs to the technical field of biomedical equipment, and particularly relates to a full-automatic RNA library preparation device applied to second-generation high-throughput sequencing.
(II) background of the invention
As one of the most important molecular biological analysis methods, the advent of sequencing technology has not only provided important data for basic biological studies such as genetic information disclosure and gene expression control, but also has played an important role in application studies such as gene diagnosis and gene therapy.
High throughput sequencing technology is a milestone in the sequencing development process, and provides unprecedented opportunities for modern life science research. The second generation high-throughput sequencing technology (second generation sequencing) can be used for genome sequencing, transcriptome sequencing, gene expression regulation, detection of transcription factor binding sites, methylation and other researches. The second generation sequencing is also the most widely and directly applied technical means for analyzing the problems of genetic mechanism of genetic diseases, pharmacogenomics, tumor personalized treatment, biomarker identification and the like at present.
In the second generation sequencing experimental procedure, the preparation of the sequencing library is a very critical step. The traditional preparation of sequencing library requires the procedures of sample nucleic acid extraction, enzyme pretreatment or mechanical shearing, linker addition for chain connection, PCR amplification and the like, and has complex operation and low automation degree. Meanwhile, in order to avoid pollution, the operations need to be performed in different laboratories, strict requirements are imposed on the physical spatial distribution of the laboratories, and operators need to be trained professionally, otherwise, the accuracy of the subsequent sequencing result is affected.
Disclosure of the invention
The invention provides a full-automatic RNA library preparation device applied to second-generation high-throughput sequencing, aiming at overcoming the defects that the automation degree of a second-generation sequencing library preparation technology in the prior art is low, pollution is easily introduced in the operation process, the accuracy of a sequencing result is influenced, the operation procedure is professional and complicated, and non-professionals cannot independently complete.
The invention is realized by the following technical scheme:
the utility model provides a be applied to full-automatic RNA library preparation facilities of second generation high-throughput sequencing, includes the box body, sets up the introduction port on the top surface of box body, and its special character lies in: the device is also provided with a library transfer port, the sample inlet and the library transfer port are provided with sealing covers which can slide left and right to seal or open the sample inlet and the library transfer port, the bottom of the box body is respectively provided with 1 library collecting pool, 1 sample pool, 9 reagent bins and 1 waste liquid tank, wherein, the sample pool is arranged opposite to the sample inlet, the library collecting pool is arranged opposite to the library transfer port, the box body is respectively provided with a screw rod and a sampling needle, the left and right ends of the screw rod are respectively connected with the corresponding sides of the box body in a rotating way through a bearing III and a bearing IV, the screw rod is horizontally arranged, the right end of the screw rod extends out of the box body to be connected with a screw rod knob, the screw rod is provided with a connecting piece which is connected with the screw rod through a thread, the top surface of the connecting piece is provided with a sampling needle accommodating hole, the central shaft of the sampling needle accommodating hole is vertical to the central shaft of the screw rod, the bottom, the first spring extends upwards from the bottom of the sampling needle containing hole and extends out of the sampling needle containing hole, the sampling needle is sleeved in the first spring, the sampling needle is communicated with the negative pressure generator through a pipeline, the negative pressure generator comprises a shell, a piston and a piston rod, a second spring is sleeved on the piston rod, the free end of the piston rod is fixedly connected with the left end of a pressure rod needle, the right end of the pressure rod needle extends out of the box body and is used for pushing the piston rod, a pressure plate is arranged above the sampling needle and comprises a pressure plate body and a pressure plate shaft connected with the pressure plate body, wherein the pressure plate shaft is arranged in parallel with the screw rod, the left end of the pressure plate shaft is rotatably connected with the box body through a bearing I, the right end of the pressure plate shaft is rotatably connected with the box body through a bearing II, and the,
the sequencing method of the full-automatic RNA library preparation device applied to the second-generation high-throughput sequencing comprises the following steps:
all reagents used for constructing the RNA library are placed in a reagent bin at the bottom of a box body in advance, an RNA sample is subjected to reverse transcription to form cDNA, DNA fragmentation, end repair, joint addition, PCR amplification and purification, the constructed library is transferred to a library collecting pool by a sampling needle, a sealing cover at the top of the box body is opened, the constructed library is taken out through a library transfer port, and then the library is placed on a sequencing platform for sequencing.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: all reagents required by library preparation are respectively placed in the reagent bins, and the open ends of the library collecting pool, the waste liquid groove and the reagent bins are sealed by tinfoil paper.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: the top in library collecting pit, sample cell, reagent storehouse, waste liquid groove sets up the perforated plate, and the perforated plate left and right-hand member sets up with the box body inner wall is fixed respectively, and the hole on the perforated plate corresponds respectively with the open end of reagent storehouse, waste liquid groove, sample cell, library collecting pit.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: the inner wall of the box body is provided with a pressing plate limiting plate for limiting the rotation angle of the pressing plate shaft, the pressing plate limiting plate is positioned below the pressing plate body, and the central shaft of the pressing plate limiting plate is perpendicular to the pressing plate shaft.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: and sealing gaskets are arranged at the mounting positions of the third bearing and the fourth bearing.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: the reagents respectively contained in the reagent bins 1-9 are a first reagent combination or a second reagent combination, wherein,
the reagent combination one is as follows:
a first reagent bin: the mixed solution of the RNA reverse transcription reagent,
a reagent bin II: DNA fragmentation reagent mixture;
a reagent bin III: EDTA solution;
and a reagent bin IV: no DNase and RNase water;
a fifth reagent bin: CMP μ re Beads;
a reagent bin six: ethanol solution;
a reagent bin seven: DNA end repair and adding A reagent mixed liquor;
and (8) reagent bin eight: connecting Adaptor with a reaction reagent mixed solution;
a reagent bin nine: the mixed solution of the PCR amplification reagent is prepared,
the reagent combination II is as follows:
a first reagent bin: the mixed solution of the RNA reverse transcription reagent,
a reagent bin II: DNA fragmentation reagent mixture;
a reagent bin III: EDTA solution;
and a reagent bin IV: nucleic-free Water;
a fifth reagent bin: AMP μ re Beads;
a reagent bin six: ethanol solution;
a reagent bin seven: DNA end repair and adding A reagent mixed liquor;
and (8) reagent bin eight: connecting Adaptor with a reaction reagent mixed solution;
a reagent bin nine: PCR amplification reagent mixture.
Wherein,
the first reagent combination is preferably:
a first reagent chamber: 4 mul of RNA reverse transcription reagent mixed solution;
a reagent chamber II: 4.2 ul of DNA fragmentation reagent mixture, main components: 10X dsDNA fragmentation B. mu.l, 100X 100 mg/ml BSA 0.2. mu.l, dsDNA fragmentation 2. mu.l;
a third reagent chamber: 10-20 μ l of 0.5M EDTA;
and a reagent chamber IV: no DNase and RNase water 310-;
a fifth reagent chamber: CMP μ re Beads 310-;
a reagent cabin six: 80% ethanol 900-;
a reagent chamber seven: DNA end repair and addition of A reagent 8.5. mu.l of mixed solution, main components: 10 XEnd repairReaction Buffer 6.5. mu.l, End Prep Enzyme Mix 2. mu.l;
and eight reagent chambers: 18.5 mu l of Adaptor ligation reaction reagent mixed solution comprises the following main components: 14 ul of T4 DNA ligasebuffer, 2 ul of T4 DNA ligase and 2.5 ul of Adaptor;
a reagent chamber nine: 27 μ l of PCR amplification reagent mixture, main components: HiFidelity 2X PCR Master Mix 25. mu.l, univocal primer 1. mu.l, Index primer 1. mu.l.
The reagent combination II is preferably:
a first reagent chamber: 4 mul of RNA reverse transcription reagent mixed solution;
a reagent chamber II: the DNA fragmentation reagent mixture is 4.2. mu.l, which is 2. mu.l of 10X dsDNA fragmentation B. mu.ffer, 0.2. mu.l of 100X 100 mg/ml BSA, 2. mu.l of dsDNA fragmentation;
a third reagent chamber: 10-20 μ l of 0.5M EDTA;
and a reagent chamber IV: nucleic-free Water 210-235. mu.l;
a fifth reagent chamber: AMP μ re Beads 215-;
a reagent cabin six: 80% ethanol 1120-;
a reagent chamber seven: DNA End Repair and adding A reagent mixed liquor 18 ul, it is NEXTflex chamber End-Repair & addition Buffer Mix 15 ul, NEXTflex chamber End-Repair & addition Enzyme Mix 3 ul;
and eight reagent chambers: 50 ul of Adaptor ligation reaction reagent mixed solution is 47.5 ul of NEXTflex ™ ligand enzyme enzemix and 2.5 ul of Adaptor;
a reagent chamber nine: PCR amplification reagent mixture 14 ul, which was NEXTflex-positive PCR Master Mix 12 ul, NEXTflex-negative Primer Mix2 ul.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: when a combination of reagents is selected, the sequencing method specifically comprises the following steps:
library preparation samples: purified single-stranded RNA, dissolved in ultrapure water, RNA concentration 150-: OD260/OD280= 1.78-2.0.
The reagent chambers 1-9 are respectively filled with RNA library preparation reagents.
The library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 10-15. mu.l of sample was pipetted into the sample cell through the injection port, which was closed.
1. Reverse transcription of RNA into cDNA
And (2) transferring 3.3-6.7 mu l of RNA sample in the sample pool to the first reagent chamber by a small liquid transfer device in the external mechanical control box body, and simultaneously transferring 9.3-12.7 mu l of DNase and RNase-free water from the fourth reagent chamber to the first reagent chamber to ensure that the total volume is 20 mu l, and reacting with the reverse transcription reagent mixed solution in the first reagent chamber under the following reaction conditions: 15min at 37 ℃; 5seconds at 85 ℃; keeping at 4 ℃.
2. DNA fragmentation reaction
And (3) mechanically operating a small-sized pipettor inside the box body from the outside to transfer 15 mu l of the cDNA solution in the first reagent cabin into the second reagent cabin, and simultaneously transferring 0.8 mu l of DNase-free and RNase-free water into the second reagent cabin from the fourth reagent cabin to ensure that the total reaction volume is 20 mu l, so that the reaction volume reacts with the DNA fragmentation reagent mixed solution in the second reagent cabin.
Reaction conditions are as follows: incubate at 35-38 ℃ for 25-30 minutes.
Then, 5. mu.l of 0.5M EDTA was aspirated from the reagent compartment three and added to the reagent compartment one to terminate the enzymatic reaction.
3. cDNA fragment purification
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10-15 times through a small-sized pipettor in the box body, and then transferring 25 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 5-10 times by a small-sized pipettor in the box body, and standing the mixture for 5-10 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a second position of the reagent chamber, and standing for 5-10 minutes to separate magnetic beads from supernatant solution;
4) a small-sized pipettor in the box body sucks the supernatant in the second reagent cabin and transfers the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 60 mul of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 30-60 seconds;
6) lifting the magnetic frame to the position of the second reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the second reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 35-40 μ l of DNase-free and RNase-free water in the reagent chamber IV into the reagent chamber I, sucking and beating the DNase-free and RNase-free water up and down by using a small-sized pipettor in the box body for 5-10 times, uniformly mixing, and standing for 5-10 minutes at room temperature;
10) and lifting the magnetic frame to a second position of the reagent chamber to separate the magnetic beads from the eluted cDNA.
4. DNA end repair reaction and A addition reaction:
1) transferring 25 mul of cDNA purified solution in the second reagent chamber to the seventh reagent chamber, then transferring 31.5 mul of DNase-free and RNase-free water in the fourth reagent chamber to the seventh reagent chamber, sucking and beating the mixture up and down for 5 to 10 times by using a small-sized pipettor in the box body, and uniformly mixing the mixture and DNA tail end repair reaction and A reaction mixed solution in the seventh reagent chamber for reaction;
2) the reaction procedure was as follows: 15-20 min at 12-15 ℃; 15-20 min at 35-37 ℃; 20-25 min at 70-72 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10-15 times through a small-sized pipettor in the box body, and then transferring 65 mu l of magnetic bead liquid into a seventh reagent chamber;
2) sucking and beating the mixture for 5-10 times by a small-sized pipettor in the box body, and standing the mixture for 5-10 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin seven, and transferring the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 30-60 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 75 μ l of DNase-free and RNase-free water in the reagent chamber IV into the reagent chamber VII, sucking and beating the DNase-free and RNase-free water in the reagent chamber IV by using a small-sized pipettor for 5-10 times, and standing the reagent chamber IV for 5-10 minutes at room temperature;
10) the magnetic frame was raised to the seventh position in the reagent chamber to separate the magnetic beads from the eluted cDNA.
6. Connection adapter
Sucking 65 mu l of cDNA purified solution in the reagent cabin seven into the reagent cabin eight, and reacting with the Adaptor connection reaction reagent mixed solution in the reagent cabin eight;
sucking and beating the mixture up and down for 5 to 10 times by using a small-sized pipettor in the box body and uniformly mixing the mixture;
reaction conditions are as follows: incubate at 20-25 ℃ for 15-20 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10-15 times through a small-sized pipettor in the box body, and then transferring 83.5 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
3) lifting the magnetic frame to the eighth position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 30-60 seconds;
6) lifting the magnetic frame to the position of the reagent cabin eight, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 30 mu l of DNase-free and RNase-free water in the reagent chamber IV into the reagent chamber VIII, sucking and beating the DNase-free and RNase-free water up and down for 5-10 times by using a small-sized pipettor in the box body, and standing the reagent chamber IV for 5-10 minutes at room temperature;
10) the magnetic frame was raised to the eight positions in the reagent chamber to separate the magnetic beads from the eluted cDNA.
8. PCR amplification
And sucking 23 mu l of the cDNA selectively recovered fragment connected with the adaptor in the reagent cabin eight, transferring the cDNA selectively recovered fragment to the reagent cabin nine, and reacting with the PCR amplification reagent mixed liquor in the reagent cabin nine.
The reaction procedure was as follows: pre-denaturation at 95-98 deg.C for 30-45 s; denaturation at 95-98 deg.C for 10-15 s; annealing at 60-65 deg.C for 30-40 s; extending at 70-72 ℃ for 30-40 s; 6-10 cycles; final extension at 70-72 deg.C for 4-5 min.
9. PCR product purification
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10-15 times through a small-sized pipettor in the box body, and then transferring 50 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin nine, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin nine, and standing for 30-60 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 5-10 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 35-40 μ l of DNase-free and RNase-free water in the reagent chamber IV into the reagent chamber IV, sucking and beating the DNase-free and RNase-free water in the reagent chamber IV for 5-10 times by using a small-sized pipettor in the box body, and standing the reagent chamber IV for 5-10 minutes at room temperature;
10) and lifting the magnetic frame to a nine-position reagent chamber to separate the magnetic beads from the PCR products.
10. Removing the PCR product
And transferring 25-30 mu l of the PCR purification solution in the reagent chamber nine to a library collecting pool, and removing the PCR purification solution from the box body from the outside through a library transfer port, wherein the PCR purification solution can be taken to a sequencing platform for cluster generation and sequencing, and the RNA library can also be stored at-20 ℃.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: when the reagent combination II is selected, the sequencing method specifically comprises the following steps:
library preparation samples: purified single-stranded RNA, dissolved in ultrapure water, RNA concentration 150-: OD260/OD280= 1.78-2.0.
The reagent chambers 1-9 are respectively filled with RNA library preparation reagents.
The library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 10-15. mu.l of sample was pipetted into the sample cell through the injection port, which was closed.
1. Reverse transcription of RNA into cDNA
And a small-sized pipettor in the external mechanical control box body transfers 3.3-6.7 mu l of the RNA sample in the sample pool to the first reagent chamber, and simultaneously transfers 9.3-12.7 mu l of the nucleic-free Water from the fourth reagent chamber to the first reagent chamber, so as to ensure that the total volume is 20 mu l, and the RNA sample reacts with the reverse transcription reagent mixed liquor in the first reagent chamber under the following reaction conditions: 15min at 37 ℃; 5seconds at 85 ℃; keeping at 4 ℃.
2. DNA fragmentation reaction
And a small-sized pipettor in the box body is mechanically operated from the outside to transfer 15 mu l of the cDNA solution in the sample pool into the second reagent cabin, and simultaneously transfer 0.8 mu l of nucleic-free Water from the fourth reagent cabin into the second reagent cabin, so that the total reaction volume is 20 mu l, and the cDNA solution reacts with the DNA fragmentation reagent mixed solution in the second reagent cabin.
Reaction conditions are as follows: incubating at 35-38 deg.C for 30-40 min.
Then, 5. mu.l of 0.5M EDTA was aspirated from the reagent compartment III and added to the reagent compartment II to terminate the enzymatic reaction.
3. DNA fragment purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10-15 times up and down through a small-sized pipettor in the box body, and then transferring 35 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 5-10 times by a small-sized pipettor in the box body, and standing the mixture for 5-10 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a second position of the reagent chamber, and standing for 5-10 minutes to separate magnetic beads from supernatant solution;
4) a small-sized pipettor in the box body sucks the supernatant in the second reagent cabin and transfers the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 60 mul of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 30-60 seconds;
6) and lifting the magnetic frame to the second position of the reagent chamber, and standing for 5-10 minutes to separate the magnetic beads from the supernatant solution. Sucking the supernatant in the reagent cabin II, and transferring the supernatant to a waste liquid groove;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 30-35 μ l of clean-free Water in the reagent chamber II, sucking up and down by using a small-sized pipettor in the box body for 5-10 times, mixing uniformly, and standing for 5-10 minutes at room temperature;
10) the magnetic frame is lifted to the second position of the reagent chamber to separate the magnetic beads from the eluted cDNA,
4. DNA end repair reaction and A addition reaction:
1) transferring 20 mul of cDNA purified solution in the second reagent chamber into the seventh reagent chamber, sucking and beating the cDNA purified solution up and down for 5 to 10 times by using a small-sized pipettor in the box body, and uniformly mixing the cDNA purified solution with the DNA tail end repair reaction and the reaction mixed solution added with A in the seventh reagent chamber to react;
the reaction procedure was as follows: 18-22 min at 22-25 ℃; 20-25 min at 70-72 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10-15 times up and down through a small-sized pipettor in the box body, and then transferring 80 mu l of magnetic bead liquid into the seventh reagent chamber;
2) sucking and beating the mixture for 5-10 times by a small-sized pipettor in the box body, and standing the mixture for 5-10 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin seven, and transferring the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 30-60 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 60 μ l of clean-free Water in the reagent chamber four into the reagent chamber seven, sucking and beating 5-10 times by using a small-sized pipette, and standing for 5-10 minutes at room temperature;
the magnetic frame is lifted to the seventh position of the reagent chamber to separate the magnetic beads from the eluted DNA.
6. Connection adapter
1) Sucking 50 mu l of cDNA purified solution in the reagent cabin seven into the reagent cabin eight, and reacting with the Adaptor connection reaction reagent mixed solution in the reagent cabin eight;
2) and (4) sucking and beating the mixture up and down for 10 to 15 times by using a small-sized pipettor in the box body and uniformly mixing the mixture.
3) Reaction conditions are as follows: incubate at 22-25 ℃ for 15-20 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber and the eighth reagent chamber for 10-15 times by a small-sized pipettor in the box body, and then transferring 60 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
3) lifting the magnetic frame to the eighth position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 30-60 seconds;
6) lifting the magnetic frame to the position of the reagent cabin eight, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 5-10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic rack, sucking 30 mu l of clean-free Water in the reagent chamber four into the reagent chamber eight, sucking and sucking for 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
10) the magnetic frame was raised to the eight positions in the reagent chamber to separate the magnetic beads from the eluted cDNA.
8. PCR amplification
And sucking 20 mu l of cDNA selectively recovered fragments after the connection of the adaptor in the reagent cabin eight, transferring the cDNA selectively recovered fragments into the reagent cabin nine, and reacting with the mixed solution of the PCR amplification reagent in the reagent cabin nine.
The reaction procedure was as follows: pre-denaturation at 95-98 deg.C for 2-2.5 min; denaturation at 95-98 deg.C for 30-40 s; annealing at 60-65 deg.C for 30-40 s; extending at 70-72 ℃ for 60-90 s; 4-10 cycles; final extension at 70-72 deg.C for 4-5 min.
9. PCR product purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10-15 times up and down through a small-sized pipettor in the box body, and then transferring 40 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin nine, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mul of 80% ethanol in the reagent cabin six to the reagent cabin nine, and standing for 30-60 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 5-10 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 5-10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic rack, sucking 35-40 μ l of clean-free Water in the reagent chamber four into the reagent chamber nine, sucking up and down 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
10) the magnetic frame is lifted to the nine positions of the reagent chamber to separate the magnetic beads from the PCR products,
10. removing the PCR product
And transferring 25-30 mu l of the PCR purification solution in the reagent chamber nine to a library collecting pool, and removing the PCR purification solution from the box body from the outside through a library transfer hole, wherein the PCR purification solution can be taken to a sequencing platform for cluster generation and sequencing, and the RNA library can also be stored at-20 ℃.
The full-automatic RNA library preparation device applied to the second generation high-throughput sequencing is characterized in that: the library preparation method specifically further comprises the steps of:
and (3) detecting the quality of the library: the prepared RNA library is subjected to agarose gel electrophoresis to detect the fragment length distribution range in the RNA library, and if the constructed library has good quality, the sequencing can be directly carried out.
The invention has the beneficial effects that: according to the invention, all reagents used for constructing the RNA library are placed in a reagent bin at the bottom of a box body in advance, an RNA sample is subjected to reverse transcription to form cDNA, DNA fragmentation, end repair, joint addition, PCR amplification and purification, the constructed library is transferred to a library collecting pool by a sampling needle, a sealing cover at the top of the box body is opened, the constructed library is taken out through a library transfer port, and then the library can be placed on a sequencing platform for subsequent sequencing. The RNA sample is added into the sample pool through the sample inlet and then the sample inlet is sealed, waste liquid and the like generated in the construction process are stored in the waste liquid tank at the bottom of the box body, the whole library construction process is completed inside the box body, the whole process is closed, the whole process is not contacted with the outside, no pollution is generated, the problem of very headache pollution of people in scientific research and clinical test work is solved, meanwhile, the manpower is liberated, even ordinary technicians in the field can operate and implement independently, and the efficiency is improved.
(IV) description of the drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic diagram of the structure of the apparatus of the present invention;
FIG. 2 is a schematic diagram of the position relationship between a pressing plate and a pressing plate limiting plate according to the present invention;
FIG. 3 is a schematic view showing the positional relationship between the screw and the connecting member according to the present invention,
in the figure, 1 box body, 11 sample inlet, 2 platen shaft, 21 bearing I, 23 bearing II, 24 platen limit plate, 25 platen body, 26 platen shaft, 3 sampling needle, 31 piston, 32 negative pressure generator, 33 second spring, 34 platen needle, 35 first spring, 36 pipeline, 4 screw, 41 bearing III, 42 bearing IV, 44 connecting piece, 5 sample cell, 61 reagent bin I, 62 reagent bin II, 63 reagent bin III, 64 reagent bin IV, 65 reagent bin V, 66 reagent bin VI, 67 reagent bin VII, 68 reagent bin VIII, 69 reagent bin VII, 7 waste liquid tank, 8 tin foil, 9 porous plate, 12 library transfer port, 13 library collecting tank.
(V) detailed description of the preferred embodiments
The embodiment is applied to full-automatic RNA library preparation facilities of second generation high-throughput sequencing and includes box body 1, sets up injection port 11 on the top surface of box body 1, its characterized in that: also provided with a library transfer port 12, the sample inlet 11 and the library transfer port 12 are provided with sealing covers which can slide left and right to close or open the sample inlet 11 and the library transfer port 12, the bottom of the box body 1 is sequentially provided with 1 library collecting pool 13, 1 sample pool 5, 8 reagent bins 61-69, 1 waste liquid tank 7, a first reagent bin 61, a second reagent bin 62, a third reagent bin 63, a fourth reagent bin 64, a fifth reagent bin 65, a sixth reagent bin 66, a seventh reagent bin 67, a eighth reagent bin 68, a ninth reagent bin 69, the sample pool 5, the library collecting pool 13 and the waste liquid tank 7 from left to right, the tops of the reagent bins 61-69 are positioned on the same plane, all reagents required by library preparation are arranged in the reagent bins 61-69, to avoid contamination of the reagents, the open ends of the library collection well 13, the waste reservoir 7, and the reagent compartments 61-69 are closed by the tinfoil 8, and the open end of the sample well 5 is not closed. The sample pool 5 is arranged opposite to the sample inlet 11, and a liquid transfer device can be used for adding the library building sample into the sample pool 5 through the sample inlet 11; the library collecting pool 13 is arranged opposite to the library transferring port 12 and is used for containing a finally constructed library, and the prepared library can be sucked out and stored through the library transferring port 12 by a liquid moving machine or directly used for sequencing; the waste liquid tank 7 is used for holding all waste liquid generated in the library preparation process.
The upper parts of the first reagent bin 61, the second reagent bin 62, the third reagent bin 63, the fourth reagent bin 64, the fifth reagent bin 65, the sixth reagent bin 66, the seventh reagent bin 67, the eighth reagent bin 68, the ninth reagent bin 69, the waste liquid groove 7, the sample pool 5 and the library collecting pool 13 are provided with a porous plate 9, the porous plate 9 is fixedly connected with the inner wall of the box body 1, and holes in the porous plate 9 respectively correspond to the open ends of the first reagent bin 61, the second reagent bin 62, the third reagent bin 63, the fourth reagent bin 64, the fifth reagent bin 65, the sixth reagent bin 66, the seventh reagent bin 67, the eighth reagent bin 68, the ninth reagent bin 69, the waste liquid groove 7, the sample pool 5 and the library collecting pool 13. The box body 1 is internally provided with a screw rod 4 and a sampling needle 3 respectively, the left end and the right end of the screw rod 4 are rotatably connected with the corresponding side of the box body 1 through a bearing III 41 and a bearing IV 42 respectively, the screw rod 4 is horizontally arranged, the right end of the screw rod 4 extends out of the box body 1 to be connected with a screw rod knob, the screw rod 4 is provided with a connecting piece 44, the connecting piece 44 is connected with the screw rod 4 through threads, the screw rod knob is rotated, the connecting piece 44 and the screw rod 4 move relatively, the top surface of the connecting piece 44 is provided with a sampling needle containing hole 45, the central shaft of the sampling needle containing hole 45 is vertical to the central shaft of the screw rod 4, the bottom of the sampling needle containing hole 45 is fixedly provided with a first spring 35, the first spring 35 is arranged through the sampling needle containing hole 45, the first spring 35 extends upwards from the bottom of the sampling needle containing hole 35 and extends out of, rotating a screw knob, moving a sampling needle between a reagent bin 61-69, a sample cell 5, a waste liquid groove 7 and a library collecting pool 13 along with a connecting piece 44, communicating the sampling needle 3 with a negative pressure generator 32 through a pipeline 36, wherein the negative pressure generator 32 comprises a shell, a piston 31 and a piston rod, a second spring 33 is sleeved on the piston rod, the free end of the piston rod is fixedly connected with the left end of a pressure rod needle 34, the right end of the pressure rod needle 34 extends out of the box body 1 and is used for pushing the piston rod, when the pressure rod needle 34 is not pushed, the second spring 33 rebounds the pressure rod needle 34 to the original position, a pressure plate 2 is arranged above the sampling needle 3, the pressure plate 2 comprises a pressure plate body 25 and a pressure plate shaft 26 connected with the pressure plate body 25, wherein the pressure plate shaft 26 is arranged in parallel with the screw rod 4, and the left end of the pressure, the right end of the pressing plate shaft 26 is rotatably connected with the box body 1 through a second bearing 23, and the right end of the pressing plate shaft 26 extends out of the box body 1 and is rotatably connected with the pressing plate. The clamp plate body 25 is used for pressing down the sampling needle 3, and the clamp plate knob is rotated, and the clamp plate shaft 26 drives the clamp plate body 25 to press down the sampling needle 3, and when the clamp plate knob is not rotated, the first spring 35 rebounds the clamp plate body 25 to the original position. Therefore, the sampling needle 3 in the box body can move left and right and up and down between different reagent chambers 61-69 at the bottom of the box body under the action of the piston 31, the negative pressure generator 32, the second spring 33 and the first spring 35, and the suction and release of liquid are completed. The reagent bins 61-69 can be sequentially added with various reagents required by reverse transcription, DNA fragmentation, fragment end repair, joint addition, PCR amplification and purification, and then sealed by tinfoil 8, so that the whole closure and zero pollution of the library preparation process are realized. The reagent placing in each reagent bin is as follows in sequence: reagent storehouse one 61 (holds reverse transcription reagent and mixes liquid), reagent storehouse two 62 (holds DNA fragmentation reagent and mixes liquid), reagent storehouse three 63 (the EDTA of splendid attire 0.5M), reagent storehouse four 64 (the splendid attire does not have DNase, RNase water), reagent storehouse five 65 (holds DNA fragment selectivity and retrieves the magnetic bead), reagent storehouse six 66 (the splendid attire 80% ethanol), reagent storehouse seven 67 (hold DNA end repair and add A reagent and mix liquid), reagent storehouse eight 68 (hold Adaptor connection reaction reagent and mix liquid), reagent storehouse nine 69 (hold PCR amplification reagent and mix liquid).
The inner wall of the box body 1 is also fixedly provided with a pressing plate limiting plate 24 for limiting the rotation angle of the pressing plate shaft 26, the pressing plate limiting plate 24 is positioned below the pressing plate body 25, the central shaft of the pressing plate limiting plate 24 is perpendicular to the pressing plate shaft 26, when the pressing plate shaft 26 rotates to a certain angle, the pressing plate 25 is in contact with the pressing plate limiting plate 24, and the pressing plate shaft 26 cannot continue to rotate.
The full-automatic library preparation device is matched with an external mechanical part for use, the external machine is controlled by a control system, instructions can be input on the control system, and liquid taking intervals, reagent in which reagent bin is taken, the volume of the reagent to be sucked and the like are set according to detection steps. Injecting a sample into the sample cell 5 through the sample inlet 11, and sealing the sample inlet; the external machine operates the internal sampling needle 3 to move left and right and up and down among different reagent bins according to the set steps, so as to complete the absorption and release of liquid and complete a series of processes of library preparation. The final prepared library is collected in a library collection well 13, and the prepared library is aspirated manually using a pipette, stored or directly sequenced.
Example 1
The sequencing steps of the full-automatic RNA library preparation device applied to the second-generation high-throughput sequencing in the embodiment are as follows:
library preparation samples: purified single-stranded RNA, dissolved in ultrapure water. RNA concentration 215 ng/. mu.l, RNA purity: OD260/OD280= 1.85.
The reagents and the volumes respectively contained in the reagent chambers 1-9 are as follows:
a first reagent chamber: RNA reverse transcription reagent mixture 4. mu.l (5 × PrimeScript RT Master Mix (Perfect read Time);
a reagent chamber II: DNA fragmentation reagent mixture 4.2 ul (main components: 10X dsDNA fragmentation reaction B. mu.ffer 2. mu.l, 100X BSA (100 mg/ml) 0.2. mu.l, dsDNA fragmentation 2. mu.l);
a third reagent chamber: 10. mu.l of 0.5M EDTA;
and a reagent chamber IV: 310 mul of DNase-free and RNase-free water;
a fifth reagent chamber: CMP μ re Beads 310 μ l;
a reagent cabin six: 900 μ l of 80% ethanol;
a reagent chamber seven: DNA End repair and addition of A reagent 8.5. mu.l (Main component: 10 XEnd repair reaction Buffer 6.5. mu.l, End Prep Enzyme Mix 2. mu.l)
And eight reagent chambers: 18.5 ul of the Adaptor ligation reaction reagent mixture (the main components are 14 ul of T4 DNA ligasebuffer, 2 ul of T4 DNA ligase and 2.5 ul of Adaptor);
a reagent chamber nine: mu.l of PCR amplification reagent mixture (main components: HiFidelity 2X PCR Master Mix 25. mu.l, univocal primer 1. mu.l, and Index primer 1. mu.l).
The library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 10. mu.l of sample was pipetted through the sample port into the sample cell and the sample port was closed. The box body was quickly swung down by hand to ensure that all reagents were concentrated at the bottom of the reagent well. (all reactions were carried out by controlling the external machine with the software program of the control system, below.)
1. Reverse transcription of RNA into cDNA
And (2) transferring 4.6 mu l of RNA sample in the sample pool to the first reagent chamber by mechanically operating a small pipette in the box body from the outside, simultaneously transferring 11.4 mu l of DNase and RNase-free water to the first reagent chamber from the fourth reagent chamber to ensure that the total volume is 20 mu l, and reacting with the reverse transcription reagent mixed solution in the first reagent chamber under the following reaction conditions: 15min at 37 ℃; 5seconds at 85 ℃; keeping at 4 ℃.
2. DNA fragmentation reaction
And (3) mechanically operating a small pipette inside the box body from the outside to transfer 15 mu l of the cDNA solution in the first reagent cabin into the second reagent cabin, and simultaneously transferring 0.8 mu l of DNase-free and RNase-free water into the second reagent cabin from the fourth reagent cabin (so that the total reaction volume is 20 mu l) to react with the DNA fragmentation reagent mixed solution in the second reagent cabin.
Reaction conditions are as follows: incubate at 37 ℃ for 30 minutes.
Then, 5. mu.l of 0.5M EDTA was aspirated from the reagent compartment three and added to the reagent compartment one to terminate the enzymatic reaction.
3. DNA fragment purification
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10 times through a small-sized pipettor in the box body, and then transferring 25 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 5 times by a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a two-position of a reagent cabin, and standing for 10 minutes to separate magnetic beads from supernatant solution;
4) a small-sized pipettor in the box body sucks the supernatant in the second reagent cabin and transfers the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 30 seconds;
6) and lifting the magnetic frame to the second position of the reagent chamber, and standing for 5 minutes to separate the magnetic beads from the supernatant solution. Sucking the supernatant in the reagent cabin II, and transferring the supernatant to a waste liquid groove;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 35 mu l of DNase-free and RNase-free water in the reagent chamber four into the reagent chamber one, sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body, uniformly mixing the mixture, and standing the mixture for 10 minutes at room temperature;
10) and lifting the magnetic frame to a second position of the reagent chamber to separate the magnetic beads from the eluted cDNA.
4. DNA end repair reaction and A addition reaction:
1) transferring 25 mul of cDNA purified solution in the second reagent chamber to the seventh reagent chamber, then transferring 31.5 mul of DNase-free and RNase-free water in the fourth reagent chamber to the seventh reagent chamber, sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body, and uniformly mixing the mixture with DNA tail end repair reaction and A reaction mixed solution in the seventh reagent chamber for reaction;
2) the reaction procedure was as follows: 20 min at 12 ℃; 20 min at 35 ℃; 25 min at 70 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10 times through a small-sized pipettor in the box body, and then transferring 65 mu l of magnetic bead liquid into a seventh reagent chamber;
2) sucking and beating the mixture for 5 times by a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, standing for 5 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin seven, and transferring the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 30 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 5 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 75 mu l of DNase-free and RNase-free water in the reagent chamber four into the reagent chamber seven, sucking and beating the DNase-free and RNase-free water up and down for 5 times by using a small-sized pipettor, and standing the reagent chamber for 10 minutes at room temperature;
10) the magnetic frame was raised to the seventh position in the reagent chamber to separate the magnetic beads from the eluted cDNA.
6. Connection adapter
1) Sucking 65 mu l of cDNA purified solution in the reagent cabin seven into the reagent cabin eight, and reacting with the Adaptor connection reaction reagent mixed solution in the reagent cabin eight;
2) sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body and uniformly mixing the mixture;
reaction conditions are as follows: incubate at 20 ℃ for 20 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10 times through a small-sized pipettor in the box body, and then transferring 83.5 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) lifting the magnetic frame to the eighth position of the reagent chamber, standing for 5 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 30 seconds;
6) lifting the magnetic frame to the position of the reagent cabin eight, standing for 5 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 30 mu l of DNase-free and RNase-free water in the reagent chamber four to the reagent chamber eight, sucking and beating the DNase-free and RNase-free water up and down for 5 times by using a small-sized pipettor in the box body, and standing the reagent chamber eight for 10 minutes at room temperature;
10) the magnetic frame was raised to the eight positions in the reagent chamber to separate the magnetic beads from the eluted cDNA.
8. PCR amplification
And sucking 23 mu l of the cDNA selectively recovered fragment connected with the adaptor in the reagent cabin eight, transferring the cDNA selectively recovered fragment to the reagent cabin nine, and reacting with the PCR amplification reagent mixed liquor in the reagent cabin nine.
The reaction procedure was as follows: pre-denaturation at 95 ℃ for 45 s; denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 40 s; extension at 70 ℃ for 40 s; 6 cycles of the reaction; final extension at 70 ℃ for 5 min.
9. PCR product purification
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 10-15 times through a small-sized pipettor in the box body, and then transferring 50 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, and standing for 5 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin nine, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin nine, and standing for 30 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 5 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 35 mu l of DNase-free and RNase-free water in the reagent chamber IV into the reagent chamber IV, sucking and beating the DNase-free and RNase-free water in the reagent chamber IV by using a small-sized pipettor in the box body up and down for 5 times, and standing the reagent chamber IV for 10 minutes at room temperature;
10) and lifting the magnetic frame to a nine-position reagent chamber to separate the magnetic beads from the PCR products.
10. Removing the PCR product
Transferring 30 mu l of the PCR purification solution in the reagent chamber nine to a library collecting pool, and moving the PCR purification solution out of the box body from the outside through a library transfer port, wherein the PCR purification solution can be taken to a sequencing platform for cluster generation and sequencing, and the cDNA library can also be stored at-20 ℃.
11. Library quality detection
The prepared RNA library is subjected to agarose gel electrophoresis to detect the fragment length distribution range in the RNA library, and if the constructed library has good quality, the sequencing can be directly carried out.
Example 2
The sequencing steps of the full-automatic RNA library preparation device applied to the second-generation high-throughput sequencing in the embodiment are as follows:
library preparation samples: purified single-stranded RNA, dissolved in ultrapure water. RNA concentration 150 ng/. mu.l, RNA purity: OD260/OD280= 2.0.
A third reagent chamber: 20. mu.l of 0.5M EDTA;
and a reagent chamber IV: 350 mul of DNase-free and RNase-free water;
a fifth reagent chamber: CMP μ re Beads 350 μ l;
a reagent cabin six: 1100 μ l of 80% ethanol;
the library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 15. mu.l of sample was pipetted through the sample port into the cuvette and the sample port was closed.
1. Reverse transcription of RNA into cDNA
And a small-sized pipettor in the box body is mechanically controlled by the external machine to transfer 6.7 mu l of RNA sample in the sample pool to the first reagent chamber, and simultaneously transfer 9.3 mu l of DNase-free and RNase-free water from the fourth reagent chamber to the first reagent chamber, so that the total volume is ensured to be 20 mu l, and the RNA sample reacts with the reverse transcription reagent mixed solution in the first reagent chamber.
2. DNA fragmentation reaction
Reaction conditions are as follows: incubate at 38 ℃ for 25 minutes.
3. DNA fragment purification
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 15 times through a small-sized pipettor in the box body, and then transferring 25 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 10 times by a small-sized pipettor in the box body up and down, and standing the mixture for 5 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a two-position of a reagent cabin, and standing for 10 minutes to separate magnetic beads from supernatant solution;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 60 seconds;
6) and lifting the magnetic frame to the position of the reagent chamber, and standing for 10 minutes to separate the magnetic beads from the supernatant solution. Sucking the supernatant in the reagent cabin II, and transferring the supernatant to a waste liquid groove;
8) standing for 15 minutes at room temperature to dry the magnetic beads in the air;
9) and lowering the magnetic frame, sucking 40 mu l of DNase-free and RNase-free water in the reagent cabin IV into the reagent cabin I, sucking and beating the mixture up and down for 10 times by using a small-sized pipettor in the box body, uniformly mixing the mixture, and standing the mixture for 5 minutes at room temperature.
4. DNA end repair reaction and A addition reaction:
1) transferring 25 mul of cDNA purified solution in the second reagent chamber to the seventh reagent chamber, then transferring 31.5 mul of DNase-free and RNase-free water in the fourth reagent chamber to the seventh reagent chamber, sucking and beating the mixture up and down for 10 times by using a small-sized pipettor in the box body, and uniformly mixing the mixture with DNA tail end repair reaction and A reaction mixed solution in the seventh reagent chamber for reaction;
2) the reaction procedure was as follows: 15min at 15 ℃; 15min at 37 ℃; 20 min at 72 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 15 times through a small-sized pipettor in the box body, and then transferring 65 mu l of magnetic bead liquid into a seventh reagent chamber;
2) sucking and beating the mixture for 10 times by a small-sized pipettor in the box body up and down, and standing the mixture for 5 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, and standing for 10 minutes to separate the magnetic beads from the supernatant solution;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 60 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
8) standing for 15 minutes at room temperature to dry the magnetic beads in the air;
9) and (3) lowering the magnetic frame, sucking 75 mu l of DNase-free and RNase-free water in the reagent chamber four into the reagent chamber seven, sucking and beating the DNase-free and RNase-free water up and down for 10 times by using a small-sized pipettor, and standing the reagent chamber four for 5 minutes at room temperature.
6. Connection adapter
Sucking and beating the mixture up and down for 10 times by using a small-sized pipettor in the box body and uniformly mixing the mixture;
reaction conditions are as follows: incubate at 25 ℃ for 15 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 15 times through a small-sized pipettor in the box body, and then transferring 83.5 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating for 10 times up and down by using a small-sized pipettor in the box body, and standing for 5 minutes at room temperature;
3) lifting the magnetic frame to eight positions of the reagent chamber, standing for 10 minutes to separate the magnetic beads from the supernatant solution;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 60 seconds;
6) lifting the magnetic frame to eight positions of the reagent cabin, standing for 10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
8) standing for 15 minutes at room temperature to dry the magnetic beads in the air;
9) and lowering the magnetic frame, sucking 30 mu l of DNase-free and RNase-free water in the reagent chamber four to the reagent chamber eight, sucking and beating the DNase-free and RNase-free water up and down for 10 times by using a small-sized pipettor in the box body, and standing the mixture for 5 minutes at room temperature.
8. PCR amplification
The reaction procedure was as follows: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10 s; annealing at 65 ℃ for 30 s; extension at 72 ℃ for 30 s; 10 cycles; final extension at 72 ℃ for 4 min.
9. PCR product purification
1) Thoroughly mixing the CMPure magnetic beads in the fifth reagent chamber by sucking and beating the CMPure magnetic beads up and down for 15 times through a small-sized pipettor in the box body, and then transferring 50 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating for 10 times up and down by using a small-sized pipettor in the box body, and standing for 5 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, and standing for 10 minutes to separate the magnetic beads from the supernatant solution;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin nine, and standing for 60 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 10 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
9) and lowering the magnetic frame, sucking 40 mu l of DNase-free and RNase-free water in the reagent chamber four to the reagent chamber nine, sucking and beating the DNase-free and RNase-free water up and down for 10 times by using a small-sized pipettor in the box body, and standing the mixture for 5 minutes at room temperature.
10. Removing the PCR product
Transferring 30 mu l of the PCR purification solution in the reagent chamber nine to a library collecting pool, and moving the PCR purification solution out of the box body from the outside through a library transfer port, wherein the PCR purification solution can be taken to a sequencing platform for cluster generation and sequencing, and the cDNA library can also be stored at-20 ℃.
The rest is the same as in example 1.
Example 3
The sequencing steps of the full-automatic RNA library preparation device applied to the second-generation high-throughput sequencing in the embodiment are as follows:
library preparation samples: purified single-stranded RNA, dissolved in ultrapure water. RNA concentration 300 ng/. mu.l, RNA purity: OD260/OD280= 1.78.
A third reagent chamber: 15 μ l of 0.5M EDTA;
and a reagent chamber IV: no DNase and RNase water 330 μ l;
a fifth reagent chamber: CMP μ re Beads 330 μ l;
a reagent cabin six: 1000 μ l of 80% ethanol;
the library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 13. mu.l of sample was pipetted through the sample port into the cuvette and the sample port was closed.
1. Reverse transcription of RNA into cDNA
And a small-sized liquid transfer device in the box body is mechanically controlled by the external machine to transfer 3.3 mu l of RNA sample in the sample pool to the first reagent chamber, and simultaneously transfer 12.7 mu l of DNase-free and RNase-free water from the fourth reagent chamber to the first reagent chamber, so that the total volume is ensured to be 20 mu l, and the RNA sample reacts with the reverse transcription reagent mixed solution in the first reagent chamber.
3. DNA fragment purification
9) And lowering the magnetic frame, sucking 38 mu l of DNase-free and RNase-free water in the reagent cabin IV into the reagent cabin I, sucking up and down by using a small-sized pipettor in the box body for 8 times, uniformly mixing, and standing for 7 minutes at room temperature.
9. PCR product purification
9) And (3) lowering the magnetic frame, sucking 35 mu l of DNase-free and RNase-free water in the reagent chamber four to the reagent chamber nine, sucking and beating the DNase-free and RNase-free water up and down 8 times by using a small-sized pipettor in the box body, and standing the reagent chamber for 8 minutes at room temperature.
10. Removing the PCR product
Transferring 28 mul of PCR purification solution in the reagent chamber nine to a library collecting pool, and moving the PCR purification solution out of the box body from the outside through a library transfer port, wherein the PCR purification solution can be taken to a sequencing platform for cluster generation and sequencing, and the cDNA library can also be stored at-20 ℃.
The rest is the same as in example 1.
Example 4
Library preparation samples: purified single-stranded RNA, dissolved in ultrapure water. RNA concentration 150 ng/. mu.l, RNA purity: OD260/OD280= 2.0.
The reagents and the volumes respectively contained in the reagent chambers 1-9 are as follows:
a first reagent chamber: RNA reverse transcription reagent mixture 4. mu.l (5 × PrimeScript RT Master Mix (Perfect read Time);
a reagent chamber II: DNA fragmentation reagent mixture 4.2. mu.l (10X dsDNA fragmentation Reaction B. mu.l, 100X BSA (100 mg/ml) 0.2. mu.l, dsDNA fragmentation 2. mu.l);
a third reagent chamber: 10. mu.l of 0.5M EDTA;
and a reagent chamber IV: nuclean-free Water 210. mu.l;
a fifth reagent chamber: AMP μ re Beads 215 μ l;
a reagent cabin six: 1120 μ l of 80% ethanol;
a reagent chamber seven: DNA End Repair and addition of A reagent mixture 18 ul (NEXTflex chamber End-Repair & amplification Buffer Mix 15 ul, NEXTflex chamber End-Repair & amplification Enzyme Mix 3 ul);
and eight reagent chambers: adaptor ligation reaction reagent mixture 50 ul (NEXTflex. RTM. Ligase Enzyme mix47.5 ul, Adaptor 2.5 ul);
a reagent chamber nine: PCR amplification reagent mixture 14 ul (NEXTflex-Mass. PCR Master Mix 12 ul, NEXTflex-Mass. Primer Mix2 ul).
The library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 10. mu.l of sample was pipetted through the sample port into the sample cell and the sample port was closed. The box body was quickly swung down by hand to ensure that all reagents were concentrated at the bottom of the reagent well. (all reactions were carried out by controlling the external machine with the software program of the control system, below.)
1. Reverse transcription of RNA into cDNA
And a small-sized pipettor in the external mechanical control box body transfers 6.7 mu l of RNA sample in the sample pool to the first reagent chamber, and simultaneously transfers 9.3 mu l of nucleic-free Water from the fourth reagent chamber to the first reagent chamber, so as to ensure that the total volume is 20 mu l, and the RNA sample reacts with the mixed solution of the reverse transcription reagent in the first reagent chamber under the following reaction conditions: 15min at 37 ℃; 5seconds at 85 ℃; keeping at 4 ℃.
2. DNA fragmentation reaction
A small pipette inside the external mechanical control box transfers 15 ul of cDNA solution in the sample pool to the second reagent compartment, and simultaneously transfers 0.8 ul of nucleic-free Water from the fourth reagent compartment to the second reagent compartment (so that the total reaction volume is 20 ul), and the cDNA solution reacts with the DNA fragmentation reagent mixed solution in the second reagent compartment.
Reaction conditions are as follows: incubate at 35 ℃ for 40 minutes.
Then, 5. mu.l of 0.5M EDTA was aspirated from the reagent compartment III and added to the reagent compartment II to terminate the enzymatic reaction.
3. DNA fragment purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10 times up and down through a small-sized pipettor in the box body, and then transferring 35 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 5 times by a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a second position of the reagent chamber, and standing for 5 minutes to separate magnetic beads from the supernatant solution;
4) a small-sized pipettor in the box body sucks the supernatant in the second reagent cabin and transfers the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 30 seconds;
6) and lifting the magnetic frame to the second position of the reagent chamber, and standing for 5 minutes to separate the magnetic beads from the supernatant solution. Sucking the supernatant in the reagent cabin II, and transferring the supernatant to a waste liquid groove;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 30 mu l of clean-free Water in the reagent chamber four into the reagent chamber two, sucking and pumping for 5 times by using a small-sized pipettor in the box body, uniformly mixing, and standing for 10 minutes at room temperature;
10) and lifting the magnetic frame to a second position of the reagent chamber to separate the magnetic beads from the eluted cDNA.
4. DNA end repair reaction and A addition reaction:
1) transferring 20 mul of cDNA purified solution in the second reagent chamber into the seventh reagent chamber, sucking and beating the cDNA purified solution up and down for 5 times by using a small-sized pipettor in the box body, and uniformly mixing the cDNA purified solution with the DNA tail end repair reaction and the reaction mixed solution added with A in the seventh reagent chamber to react;
2) the reaction procedure was as follows: 22 min at 22 ℃; 25 min at 70 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10 times up and down through a small-sized pipettor in the box body, and then transferring 80 mu l of magnetic bead liquid into the seventh reagent chamber;
2) sucking and beating the mixture for 5 times by a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, standing for 5 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin seven, and transferring the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 30 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 5 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 60 μ l of clean-free Water in the reagent chamber four into the reagent chamber seven, sucking and beating for 5 times by using a small-sized pipettor, and standing for 10 minutes at room temperature;
10) the magnetic frame is lifted to the seventh position of the reagent chamber to separate the magnetic beads from the eluted DNA.
6. Connection adapter
1) Sucking 50 mu l of cDNA purified solution in the reagent cabin seven into the reagent cabin eight, and reacting with the Adaptor connection reaction reagent mixed solution in the reagent cabin eight;
2) sucking and beating the mixture up and down for 10 times by using a small-sized pipettor in the box body and uniformly mixing the mixture;
3) reaction conditions are as follows: incubate at 22 ℃ for 20 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10 times up and down through a small-sized pipettor in the box body, and then transferring 60 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) lifting the magnetic frame to the eighth position of the reagent chamber, standing for 5 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 30 seconds;
6) lifting the magnetic frame to the position of the reagent cabin eight, standing for 5 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 5 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 30 mu l of clean-free Water in the reagent chamber four into the reagent chamber eight, sucking and beating for 5 times by using a small-sized pipettor in the box body, and standing for 10 minutes at room temperature;
10) the magnetic frame was raised to the eight positions in the reagent chamber to separate the magnetic beads from the eluted cDNA.
8. PCR amplification
And sucking 20 mu l of cDNA selectively recovered fragments after the connection of the adaptor in the reagent cabin eight, transferring the cDNA selectively recovered fragments into the reagent cabin nine, and reacting with the mixed solution of the PCR amplification reagent in the reagent cabin nine.
The reaction procedure was as follows: pre-denaturation at 95 ℃ for 2.5 min; denaturation at 95 ℃ for 40 s; annealing at 60 ℃ for 40 s; extension at 70 ℃ for 90 s; 4 cycles; final extension at 70 ℃ for 5 min.
9. PCR product purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10 times up and down through a small-sized pipettor in the box body, and then transferring 40 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating the mixture up and down for 5 times by using a small-sized pipettor in the box body, and standing the mixture for 10 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, and standing for 5 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin nine, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mu l of 80% ethanol in the reagent cabin VI to the reagent cabin ninth, and standing for 30 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 5 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 5 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 35 μ l of clean-free Water in the reagent chamber four into the reagent chamber nine, sucking and sucking for 5 times by using a small-sized pipettor in the box body, and standing for 10 minutes at room temperature;
10) and lifting the magnetic frame to a nine-position reagent chamber to separate the magnetic beads from the PCR products.
10. Removing the PCR product
Transferring 25 mu l of the PCR purification solution in the reagent chamber nine to a library collecting pool, and moving the PCR purification solution out of the box body from the outside through a library transfer hole, wherein the PCR purification solution can be taken to a sequencing platform for cluster generation and sequencing, and the RNA library can also be stored at-20 ℃.
11. Library quality detection
The prepared RNA library is subjected to agarose gel electrophoresis to detect the fragment length distribution range in the RNA library, and if the constructed library has good quality, the sequencing can be directly carried out.
Example 5
Library preparation samples: purified single-stranded RNA, dissolved in ultrapure water. RNA concentration 300 ng/. mu.l, RNA purity: OD260/OD280= 1.78.
The reagents and the volumes respectively contained in the reagent chambers 1-9 are as follows:
a first reagent chamber: RNA reverse transcription reagent mixture 4. mu.l (5 × PrimeScript RT Master Mix (Perfect read Time);
a reagent chamber II: DNA fragmentation reagent mixture 4.2. mu.l (10X dsDNA fragmentation Reaction B. mu.l, 100X BSA (100 mg/ml) 0.2. mu.l, dsDNA fragmentation 2. mu.l);
a third reagent chamber: 10-20 μ l of 0.5M EDTA;
and a reagent chamber IV: nucleic-free Water 210-235. mu.l;
a fifth reagent chamber: AMP μ re Beads 215-;
a reagent cabin six: 80% ethanol 1120-;
a reagent chamber seven: DNA End Repair and addition of A reagent mixture 18 ul (NEXTflex chamber End-Repair & amplification Buffer Mix 15 ul, NEXTflex chamber End-Repair & amplification Enzyme Mix 3 ul);
and eight reagent chambers: adaptor ligation reaction reagent mixture 50 ul (NEXTflex. RTM. Ligase Enzyme mix47.5 ul, Adaptor 2.5 ul);
a reagent chamber nine: PCR amplification reagent mixture 14 ul (NEXTflex-Mass. PCR Master Mix 12 ul, NEXTflex-Mass. Primer Mix2 ul).
The library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 10-15. mu.l of sample was pipetted into the sample cell through the injection port, which was closed. The box body was quickly swung down by hand to ensure that all reagents were concentrated at the bottom of the reagent well. (all reactions were carried out by controlling the external machine with the software program of the control system, below.)
1. Reverse transcription of RNA into cDNA
And a small-sized pipettor in the external mechanical control box body transfers 3.3-6.7 mu l of the RNA sample in the sample pool to the first reagent chamber, and simultaneously transfers 9.3-12.7 mu l of the nucleic-free Water from the fourth reagent chamber to the first reagent chamber, so as to ensure that the total volume is 20 mu l, and the RNA sample reacts with the reverse transcription reagent mixed liquor in the first reagent chamber under the following reaction conditions: 15min at 37 ℃; 5seconds at 85 ℃; keeping at 4 ℃.
2. DNA fragmentation reaction
A small pipette inside the external mechanical control box transfers 15 ul of cDNA solution in the sample pool to the second reagent compartment, and simultaneously transfers 0.8 ul of nucleic-free Water from the fourth reagent compartment to the second reagent compartment (so that the total reaction volume is 20 ul), and the cDNA solution reacts with the DNA fragmentation reagent mixed solution in the second reagent compartment.
Reaction conditions are as follows: incubate at 35-38 ℃ for 30-40 minutes.
Then, 5. mu.l of 0.5M EDTA was aspirated from the reagent compartment III and added to the reagent compartment II to terminate the enzymatic reaction.
3. DNA fragment purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10-15 times up and down through a small-sized pipettor in the box body, and then transferring 35 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 5-10 times by a small-sized pipettor in the box body, and standing the mixture for 5-10 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a second position of the reagent chamber, and standing for 5-10 minutes to separate magnetic beads from supernatant solution;
4) a small-sized pipettor in the box body sucks the supernatant in the second reagent cabin and transfers the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 60 mul of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 30-60 seconds;
6) and lifting the magnetic frame to the second position of the reagent chamber, and standing for 5-10 minutes to separate the magnetic beads from the supernatant solution. Sucking the supernatant in the reagent cabin II, and transferring the supernatant to a waste liquid groove;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 30-35 μ l of clean-free Water in the reagent chamber II, sucking up and down by using a small-sized pipettor in the box body for 5-10 times, mixing uniformly, and standing for 5-10 minutes at room temperature;
10) and lifting the magnetic frame to a second position of the reagent chamber to separate the magnetic beads from the PCR products.
4. DNA end repair reaction and A addition reaction:
1) transferring 20 mul of cDNA purified solution in the second reagent chamber into the seventh reagent chamber, sucking and beating the cDNA purified solution up and down for 5 to 10 times by using a small-sized pipettor in the box body, and uniformly mixing the cDNA purified solution with the DNA tail end repair reaction and the reaction mixed solution added with A in the seventh reagent chamber to react;
2) the reaction procedure was as follows: 18-22 min at 22-25 ℃; 20-25 min at 70-72 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10-15 times up and down through a small-sized pipettor in the box body, and then transferring 80 mu l of magnetic bead liquid into the seventh reagent chamber;
2) sucking and beating the mixture for 5-10 times by a small-sized pipettor in the box body, and standing the mixture for 5-10 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin seven, and transferring the supernatant to a waste liquid groove;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 30-60 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 10-15 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 60 μ l of clean-free Water in the reagent chamber four into the reagent chamber seven, sucking and beating 5-10 times by using a small-sized pipette, and standing for 5-10 minutes at room temperature;
10) the magnetic frame is lifted to the seventh position of the reagent chamber to separate the magnetic beads from the eluted DNA.
6. Connection adapter
1) Sucking 50 mu l of cDNA purified solution in the reagent cabin seven into the reagent cabin eight, and reacting with the Adaptor connection reaction reagent mixed solution in the reagent cabin eight;
2) sucking and beating the mixture up and down for 10 to 15 times by using a small-sized pipettor in the box body and uniformly mixing the mixture;
3) reaction conditions are as follows: incubate at 22-25 ℃ for 15-20 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber and the eighth reagent chamber for 10-15 times by a small-sized pipettor in the box body, and then transferring 60 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
3) lifting the magnetic frame to the eighth position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 30-60 seconds;
6) lifting the magnetic frame to the position of the reagent cabin eight, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 5-10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic rack, sucking 30 mu l of clean-free Water in the reagent chamber four into the reagent chamber eight, sucking and sucking for 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
10) and lifting the magnetic frame to the eight positions of the reagent chamber to separate the magnetic beads from the PCR products.
8. PCR amplification
And sucking 20 mu l of cDNA selectively recovered fragments after the connection of the adaptor in the reagent cabin eight, transferring the cDNA selectively recovered fragments into the reagent cabin nine, and reacting with the mixed solution of the PCR amplification reagent in the reagent cabin nine.
The reaction procedure was as follows: pre-denaturation at 95-98 deg.C for 2-2.5 min; denaturation at 95-98 deg.C for 30-40 s; annealing at 60-65 deg.C for 30-40 s; extending at 70-72 ℃ for 60-90 s; 4-10 cycles; final extension at 70-72 deg.C for 4-5 min.
9. PCR product purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 10-15 times up and down through a small-sized pipettor in the box body, and then transferring 40 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, standing for 5-10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin nine, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mul of 80% ethanol in the reagent cabin six to the reagent cabin nine, and standing for 30-60 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 5-10 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
7) repeating the steps 5 and 6;
8) standing for 5-10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic rack, sucking 35-40 μ l of clean-free Water in the reagent chamber four into the reagent chamber nine, sucking up and down 5-10 times by using a small-sized pipettor in the box body, and standing for 5-10 minutes at room temperature;
10) and lifting the magnetic frame to a nine-position reagent chamber to separate the magnetic beads from the PCR products.
10. Removing the PCR product
Transferring 25-30 μ l of the PCR purified solution in the reagent chamber nine to a library collecting pool, and removing the PCR purified solution from the box body from the outside through a library transfer hole, wherein the PCR purified solution can be taken to a sequencing platform for cluster generation and sequencing, and the cDNA library can also be stored at-20 ℃.
11. Library quality detection
The prepared cDNA library is subjected to agarose gel electrophoresis to detect the fragment length distribution range in the cDNA library, and if the constructed library has good quality, the sequencing can be directly carried out.
Example 5
Library preparation samples: purified single-stranded RNA, dissolved in ultrapure water. RNA concentration 300 ng/. mu.l, RNA purity: OD260/OD280= 1.78.
A third reagent chamber: 20. mu.l of 0.5M EDTA;
and a reagent chamber IV: nuclear-free Water 235. mu.l;
a fifth reagent chamber: AMP μ re Beads 230 μ l;
a reagent cabin six: 1200. mu.l of 80% ethanol.
The library preparation procedure was as follows:
library preparation was performed with 1. mu.g RNA sample, and 15. mu.l of sample was pipetted through the sample port into the cuvette and the sample port was closed.
1. Reverse transcription of RNA into cDNA
A small pipette inside the external mechanical control box transfers 6.6. mu.l of RNA sample in the sample pool to the first reagent chamber, and simultaneously transfers 9.4. mu.l of nucleic-free Water from the fourth reagent chamber to the first reagent chamber, so as to ensure that the total volume is 20. mu.l.
2. DNA fragmentation reaction
Reaction conditions are as follows: incubate at 38 ℃ for 30 minutes.
3. DNA fragment purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 15 times up and down through a small-sized pipettor in the box body, and then transferring 35 mu l of magnetic bead liquid into the second reagent chamber;
2) sucking and beating the mixture for 10 times by a small-sized pipettor in the box body up and down, and standing the mixture for 5 minutes at room temperature;
3) controlling a magnetic frame device of an external machine to ascend to a two-position of a reagent cabin, and standing for 10 minutes to separate magnetic beads from supernatant solution;
5) lowering the magnetic frame, sucking 60 mu l of 80% ethanol in the reagent cabin six to the reagent cabin two, and standing for 60 seconds;
6) and lifting the magnetic frame to the position of the reagent chamber, and standing for 10 minutes to separate the magnetic beads from the supernatant solution. Sucking the supernatant in the reagent cabin II, and transferring the supernatant to a waste liquid groove;
8) standing for 15 minutes at room temperature to dry the magnetic beads in the air;
9) and lowering the magnetic frame, sucking 35 mu l of clean-free Water in the reagent chamber four into the reagent chamber two, sucking up and down by using a small-sized pipettor in the box body for 10 times, uniformly mixing, and standing for 5 minutes at room temperature.
4. DNA end repair reaction and A addition reaction:
1) transferring 20 mul of cDNA purified solution in the second reagent chamber into the seventh reagent chamber, sucking and beating the cDNA purified solution by a small-sized pipettor in the box body up and down for 10 times to uniformly mix the cDNA purified solution with the DNA tail end repair reaction in the seventh reagent chamber and the reaction mixed solution added with A to react;
2) the reaction procedure was as follows: 18 min at 25 ℃; 20 min at 72 ℃; keeping at 4 ℃.
5. Purification of end repair DNA
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 15 times up and down through a small-sized pipettor in the box body, and then transferring 80 mu l of magnetic bead liquid into the seventh reagent chamber;
2) sucking and beating the mixture for 10 times by a small-sized pipettor in the box body up and down, and standing the mixture for 5 minutes at room temperature;
3) lifting the magnetic frame to a seventh position of the reagent chamber, and standing for 10 minutes to separate the magnetic beads from the supernatant solution;
5) lowering the magnetic frame, sucking 100 mu l of 80% ethanol in the reagent cabin six to the reagent cabin seven, and standing for 60 seconds;
6) lifting the magnetic frame to the seventh position of the reagent chamber, standing for 10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the seventh reagent chamber, and transferring the supernatant to a waste liquid tank;
8) standing for 15 minutes at room temperature to dry the magnetic beads in the air;
9) the magnetic frame was lowered, 60. mu.l of clean-free Water in the reagent compartment four was pipetted into the reagent compartment seven, pipetted up and down 10 times using a small pipette, and allowed to stand at room temperature for 5 minutes.
6. Connection adapter
2) Sucking and beating the mixture up and down for 15 times by using a small-sized pipettor in the box body and uniformly mixing the mixture;
3) reaction conditions are as follows: incubate at 25 ℃ for 15 minutes.
7. Selective recovery of DNA fragments ligated to adaptor
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 15 times up and down through a small-sized pipettor in the box body, and then transferring 60 mu l of magnetic bead liquid into the eighth reagent chamber;
2) sucking and beating for 10 times up and down by using a small-sized pipettor in the box body, and standing for 5 minutes at room temperature;
3) lifting the magnetic frame to eight positions of the reagent chamber, standing for 10 minutes to separate the magnetic beads from the supernatant solution;
5) lowering the magnetic frame, sucking 200 mu l of 80% ethanol in the reagent cabin six to the reagent cabin eight, and standing for 60 seconds;
6) lifting the magnetic frame to eight positions of the reagent cabin, standing for 10 minutes to separate the magnetic beads from the supernatant solution, sucking the supernatant in the reagent cabin eight, and transferring the supernatant to a waste liquid tank;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) and lowering the magnetic frame, sucking 30 mu l of clean-free Water in the reagent chamber four into the reagent chamber eight, sucking up and down 10 times by using a small-sized pipettor in the box body, and standing for 5 minutes at room temperature.
8. PCR amplification
The reaction procedure was as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 30 s; annealing at 65 ℃ for 30 s; extension at 72 ℃ for 60 s; 10 cycles; final extension at 72 ℃ for 4 min.
9. PCR product purification
1) Thoroughly mixing AMPure magnetic beads in the fifth reagent chamber by sucking and beating the fifth reagent chamber for 15 times up and down through a small-sized pipettor in the box body, and then transferring 40 mu l of magnetic bead liquid into the ninth reagent chamber;
2) sucking and beating for 10 times up and down by using a small-sized pipettor in the box body, and standing for 5 minutes at room temperature;
3) lifting the magnetic frame to a ninth position of the reagent chamber, and standing for 10 minutes to separate the magnetic beads from the supernatant solution;
4) sucking the supernatant in the reagent cabin nine, and transferring the supernatant to a waste liquid tank;
5) lowering the magnetic frame, sucking 200 mu l of 80% ethanol in the reagent cabin VI to the reagent cabin ninth, and standing for 60 seconds;
6) lifting the magnetic frame to a ninth position of the reagent chamber to separate the magnetic beads from the supernatant solution, standing for 10 minutes, sucking the supernatant in the ninth position of the reagent chamber, and transferring the supernatant to a waste liquid tank;
8) standing for 10 minutes at room temperature to dry the magnetic beads in the air;
9) lowering the magnetic frame, sucking 40 mu l of clean-free Water in the reagent chamber four into the reagent chamber nine, sucking up and down 10 times by using a small-sized pipettor in the box body, and standing for 5 minutes at room temperature;
10) and lifting the magnetic frame to a nine-position reagent chamber to separate the magnetic beads from the PCR products.
10. Removing the PCR product
Transferring 30 mu l of the PCR purified solution in the reagent chamber nine to a library collecting pool, and moving the PCR purified cDNA solution out of the box body from the outside through a library transfer hole, wherein the PCR purified cDNA solution can be taken to a sequencing platform for cluster generation and sequencing, and the cDNA library can also be stored at-20 ℃.
The rest is the same as in example 4.