Method for amplifying or detecting full-length transcriptome cDNA containing full-length poly(A)

Abstract

The present invention discloses a method for amplifying or detecting full-length transcriptome cDNA containing full-length poly(A). The method includes: (1) single-cell reaction preparation; (2) terminal extension: using 10×barcoded gel beads with Poly(dT) as a template to extend the ends of transcripts with poly(A) to obtain single-stranded product 1; (3) template replacement: performing reverse transcription on the product 1, adding 3 bases C to the 3' end after the reverse transcription product; and adding a TSO primer for reaction to obtain product 2; (4) PCR amplification: performing PCR amplification on the product 2. The present invention improves the library construction throughput of single-cell full-length transcriptomes and fills the technical gap that current single-cell transcriptome detection cannot detect full-length poly(A).

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a method for amplifying or detecting full-length transcriptome cDNA containing full-length poly(A) and its application. Background Art

[0002] As the basis of life science research, full-length transcriptome sequencing has promoted basic and applied research in various aspects such as gene function, gene expression regulation, and evolutionary relationships of various species. At present, the research on full-length transcriptomes actually only includes the reading of partial poly(A), and cannot reflect the true information of poly(A) length. The RNA poly(A) tail is an important part of mature mRNA and lncRNA, and also has an important regulatory role in RNA stability and translation. Currently, the related technologies for poly(A) tail detection are still very limited, and next-generation sequencing cannot read homopolymer long sequences. Recently, the literature reported a new method for detecting poly(A) length based on the PacBio platform, PAIso-seq, which can obtain poly(A) length information while obtaining full-length transcriptome information.

[0003] Currently, a set of detectable poly(A) tail detection techniques, PAIso-seq, has been developed based on PacBio Iso-Seq, realizing the determination of RNA poly(A) tail sequences. Library construction process: First, terminal extension is performed with oligo dT containing U. After the extension, the oligo dT containing U is digested, and the RT primer is retained for template replacement to obtain full-length cDNA, which is amplified and then circular adapters are ligated to construct a PAIso-seq library retaining the RNA poly(A) tail for sequencing. (For the detailed library construction process, see fig.1 in https: / / www.nature.com / articles / s41467-019-13228-9#Sec11. Literature source: Liu, Y., Nie, H., Liu, H. et al. Poly(A) inclusive RNA isoform sequencing (PAIso-seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails. Nat Commun 10, 5292 (2019). https: / / doi.org / 10.1038 / s41467-019-13228-9.)

[0004] The prior art mainly focuses on the population cell level. At the single cell level, it is currently limited by the detection throughput of the method and cannot achieve high-throughput single cell transcriptome detection of full-length poly(A). The single cell full-length transcript (including full-length poly(A)) results in a long detection time and high cost. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defect in the prior art that the transcriptome containing the full-length poly(A) length cannot be detected or amplified in a high-throughput manner, and only single cells can be selected for reaction. A method for amplifying or detecting full-length transcriptome cDNA containing full-length poly(A) is provided. The method of the present invention can achieve the detection of the full-length transcriptome containing the full-length poly(A) length at the single cell level. Moreover, based on the high-throughput single cell full-length poly(A) transcriptome products of the 10X Genomics and PacBio platforms, a complete solution for more refined evolution of cell development or change is provided.

[0006] On the one hand, although the current high-throughput technology has developed relatively maturely, there is no high-throughput technology for detecting polyA. And currently, the detection tape with polyT is only used to capture mRNA and detect the mRNA information by randomly binding to any position of polyA. However, the inventor specifically utilizes the key structure on the gelbeads in the 10X Genomics platform, adjusts the master mix in the original 10X kit so that it is not limited to cDNA synthesis, and combines with the TSO technology for detection, enabling the detection of the high-throughput single cell full-length poly(A) transcriptome, and providing a complete solution for more refined evolution of cell development or change.

[0007] On the other hand, the current high-throughput (simultaneously labeling thousands of single cells for distinction) single cell sequencing needs to rely on microfluidic technology to separate single cells, and the high-throughput single cell sequencing can only detect the 3'-end sequence; while the technology that can detect the full length of polyA cannot achieve high-throughput. Therefore, this technology can perform the detection of the transcriptome containing the full length of polyA without changing the existing components, and has an irreplaceable role.

[0008] The present invention solves the above technical problems through the following technical solutions.

[0009] One of the technical solutions of the present invention is: A method for amplifying or detecting full-length transcriptome cDNA containing full-length poly(A) based on the 10X genomics single cell platform, the method comprising:

[0010] (1) Single-cell reaction preparation; it includes a) preparing a single-cell suspension; b) formulating a Master mix; c) mixing a) and b) to obtain a mixed solution and preparing droplets; wherein the Master mix includes dNTP, a DNA polymerase without 5'->3' exonuclease activity, and a buffer;

[0011] (2) Terminal extension: Using 10×barcoded gel beads with Poly(dT) as a template, extending the poly(A)-tailed transcript at the end to obtain a single-stranded product 1;

[0012] (3) Template replacement: Reverse transcribing the product 1, adding 3 bases C to the 3' end after the reverse transcription product; and adding a TSO primer for reaction to obtain product 2;

[0013] (4) PCR amplification: Performing PCR amplification on the product 2.

[0014] The buffer used in step (1) can be a conventional reverse transcription buffer. For example, it can be a buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 and 0.02 M DTT; it can also be NEB buffer 2, for example, a buffer containing 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 and 1 mM DTT.

[0015] In a preferred embodiment of the present invention, in "a) preparing a single-cell suspension" of step (1) above, every 43.2 μL of the single-cell suspension is prepared by mixing 8 μL of RNase inhibitor and a standard cell with a concentration of 700 - 1200 cells / μL, and making up with NF water; the number of the standard cells is preferably 10,000.

[0016] In a preferred embodiment of the present invention, in "b) formulating a Master mix" of step (1) above, every 31.8 μL of the Master mix contains 6.1 μL of dNTP with a concentration of 10 μM, 9.7 μL of Klenow fragment with a concentration of 50 units / μL, 14 μL of 10×NEB buffer 2, and 2 μL of reducing agent B; the buffer is 10×NEB buffer 2.

[0017] In a preferred embodiment of the present invention, "c) preparing droplets" in step (1) above is as follows: The mixed solution, Gel Beads, and Partitioning Oil are sequentially added to wells labeled 1, 2, and 3 of the GEM Chip G, respectively, and a 10× Gasket is covered on the GEM Chip G to prepare droplets.

[0018] Preferably, in the droplets, the volume ratio of the single-cell suspension, Master mix, Gel Beads, and Partitioning Oil is 43.2:31.8:50:45.

[0019] Preferably, the terminal extension in step (2) includes:

[0020] a) Reacting the product obtained in step (1) at 37 °C for 1 hour;

[0021] b) Adding a Recovery Agent to the product obtained in step a) at a volume ratio of 4:5. After the liquid layers are separated, the mixed liquid of the Recovery Agent and Partitioning Oil is removed; for example, 100 μL of the product obtained in a) and 125 μL of the Recovery Agent described in b) are used.

[0022] In addition, before step (3), it is preferred to perform sample purification and primer digestion on the product obtained in step (2).

[0023] Among them, the sample purification preferably includes:

[0024] 1) Mix Dynabeads MyOne SILANE, Cleanup Buffer, Reducing Agent B, and Nuclease-free Water to prepare Dynabeads Cleanup Mix; among them, the volume ratio of Dynabeads MyOne SILANE, Cleanup Buffer, Reducing Agent B, and Nuclease-free Water is, for example, 8:182:5:5, and the volume of Dynabeads Cleanup Mix is, for example, 200 μL;

[0025] 2) Mix the Dynabeads Cleanup Mix with the product obtained in step (2) and let it stand at room temperature; optionally, mix it by pipetting again and let it stand at room temperature; the standing time is, for example, 5 minutes;

[0026] 3) Add 80% ethanol to the Dynabeads Cleanup Mix in a volume ratio of 2:3, let it stand in the tube, and discard the supernatant; the standing time is preferably 30 seconds;

[0027] 4) Optionally, repeat step 3) once, centrifuge the centrifuge tube briefly for 2 s after discarding the supernatant, let it stand until clear, and remove the residual ethanol;

[0028] 5) Add Elution Solution I for re-dissolution; the volume ratio of Elution Solution I to the mixed solution obtained in step 3) is, for example, 1:10 - 1:15; preferably, Elution Solution I is prepared by mixing Buffer EB, 10% Tween 20, and Reducing Agent B, and the volume ratio of Buffer EB, 10% Tween 20, and Reducing Agent B is preferably 98:1:1; Reducing Agent B is, for example, from 10XGenomics;

[0029] 6) After pipetting and mixing evenly, let it stand at room temperature of 25 °C for 10 min.

[0030] Regarding primer digestion, in the prior art, usually a U is designed on the primer, and then the primer containing U is digested with User enzyme, and the digested primer is purified with magnetic beads. In the present invention, the unparticipated single-stranded primer can be directly digested with Exonuclease I and then purified.

[0031] The primer digestion in the present invention preferably includes:

[0032] 1) Add an equal amount of Exonuclease I and RNase inhibitor to the purified product tube, and then add 10× NEBbuffer 2 and place it at 37 °C for reaction; the reaction time is preferably 30 min, and the volume ratio of the purified product, Exonuclease I, RNase inhibitor, and 10X NEB buffer 2 is, for example, (30 - 35):1:1:4;

[0033] 2) Add 1.8 times the volume of room temperature RNAClean XP Beads to the PCR product and mix well, and incubate at room temperature; the incubation time is, for example, 5 min;

[0034] 3) Let it stand for, for example, 2 - 5 min until the liquid is clear, discard the supernatant; add about an equal volume of 80% ethanol, let it stand at room temperature for, for example, 30 s, and then discard the supernatant;

[0035] 4) Repeat step 3), discard the supernatant and try to suck out as much liquid at the bottom of the tube as possible;

[0036] 5) Dry at room temperature until there is no reflection and no cracking on the surface of the magnetic beads;

[0037] 6) Add TE for cDNA elution, pipette and mix well and dissolve at room temperature; the preferred dissolution time is 5 min;

[0038] 7) Place the centrifuge tube on the magnetic stand and let it stand until the liquid becomes clear, then transfer the supernatant to a new centrifuge tube; the time for the liquid to become clear is, for example, 2 - 5 min.

[0039] Preferably, superscriptTM II / IV is used for the reverse transcription in step (3).

[0040] In a preferred embodiment of the present invention, for every 50 μL of the PCR amplification system in step (4), it includes:

[0041] 9 μL of the product obtained in step (3), 25 μL of 2X KAPA HiFi HotStart ReadyMix, 2 μL of the upstream primer with a concentration of 10 μM as shown in SEQ ID NO:1, 2 μL of the downstream primer with a concentration of 10 μM as shown in SEQ ID NO:2, and 12 μL of NF water.

[0042] The second technical solution of the present invention is: A method for constructing a library of a full-length transcriptome containing poly(A), and the library construction method includes the method as described in the first technical solution.

[0043] Based on the method described in the first technical solution, preferably, the library construction method further includes end repair of the product obtained in step (4).

[0044] Preferably, the volume of the reaction system for the end repair is 30 μL; more preferably, for every 30 μL of the reaction system, it contains: 25 μL of the product, 3.5 μL of NEBNext ΜLtra II End Prep Reaction Buffer, and 1.5 μL of NEBNext ΜLtra II End Prep Enzyme Mix.

[0045] Preferably, the reaction conditions for the end repair are: React the reaction system at 20 °C for 30 min, and then at 65 °C for 30 min.

[0046] After the end repair, adapters can be added to the product, and the sequence of the adapter is, for example, as shown in SEQ ID NO:3.

[0047] Preferably, enzymatic digestion reaction and magnetic bead purification are also carried out after adding the adapter;

[0048] Preferably, in the reaction system of the enzymatic digestion, every 95 μL contains 42.5 μL of the product after adding the adapter, 10 μL of reaction buffer, 1 μL of exonuclease I with a concentration of 20 U / μL, 1 μL of exonuclease III with a concentration of 100 U / μL, and 40.5 μL of nuclease-free water.

[0049] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0050] The reagents and raw materials used in the present invention are all commercially available.

[0051] The positive and progressive effects of the present invention are as follows:

[0052] The present invention can, through end extension, label the single-cell transcriptome with molecular tags after the single-cell transcriptome is extended in a droplet environment by the single-cell platform, retain the RT primer for template replacement, obtain full-length cDNA and amplify it, and then carry out circular adapter ligation to construct a complete transcriptome library retaining the RNA poly(A) tail. The present invention improves the library construction throughput for the single-cell full-length transcriptome and makes up for the technical blank that the current single-cell transcriptome detection cannot detect the full-length poly(A). Description of the Drawings

[0053] Figure 1 It is the technical principle for high-throughput single-cell full-length transcriptome library construction containing poly(A).

[0054] Figure 2 It is the library quality inspection result of the full-length transcriptome library construction containing poly(A).

[0055] Figure 3 It is the Poly(A) length distribution. Detailed Embodiments

[0056] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0057] Example 1 Use the high-throughput single-cell full-length transcriptome library construction technology containing Poly(A) of the present invention to construct a library and sequence the breast cancer cell line 4T1 cells

[0058] The kits and reagents used mainly include Chromium Next GEM Chip G Single Cell Kit, 16rxns PN-1000127 (10X Genomics, 1000127), Chromium Next GEM Single Cell 3′GEM, Library&Gel Bead Kit v3.1 (10X Genomics, 1000128), Qubit TM RNA HS Assay Kit (Invitrogen), dsDNA BR Assay Kit or HS Assay Kit (Invitrogen), 2100 HighSensitivity DNA kit (Agilent), PB Beads (Pacific Biosciences).

[0059] The principle and process of library construction are shown in detail in Figure 1 . Specifically as follows:

[0060] 1. Single-cell Poly(A) tail extension

[0061] 1.1 Preparation of single-cell reaction

[0062] 1) According to the experimental needs, take 10,000 standard cells with a counted concentration of 700 - 1200 (cells / μL) into a labeled 0.2 mL PCR tube, add 8 μL of RNase inhibitor (Invitrogen), and then supplement NF water to a volume of 43.2 μL to prepare the cell suspension.

[0063] 2) Take another 0.2 mL PCR tube and label it as Master mix, and then prepare the reaction system as shown in Table 1 below:

[0064] Table 1

[0065] Reagent Name Single Tube Dosage (μL) dNTP (10μM, NEB) 6.1 Klenow Fragment (50units / μL NEB) 9.7 NEB buffer 2 (10X, NEB) 14 Reducing Agent B (10X Genomics) 2 total 31.8

[0066] 3) After gently mixing the cell suspension with the Master mix, take 70 μL of the mixed solution and add it to row labeled 1 of the GEM Chip G.

[0067] 4) Prepare the Gel Beads. After taking them out from -80 °C, wait for them to melt, then shake and mix for 30 seconds, briefly centrifuge for 5 seconds, and slowly aspirate 50 μL and add it to row labeled 2 of the GEM Chip G.

[0068] 5) Take 45 μL of Partitioning Oil, and label row 3 of the GEM Chip G. Cover the GEM Chip G with a 10x Gasket, and then place it in the Controller to generate microdroplets for about 18 minutes;

[0069] 6) After the microdroplets are generated, take out about 100 μL of the microdroplet generation solution and transfer it to a labeled 0.2 mL PCR tube, and place it on ice for later use.

[0070] 1.2 Terminal extension

[0071] 1) Put the product (100 μL) from the previous step into a PCR instrument and react at 37 °C for 1 hour.

[0072] 2) After the reaction is completed, add 125 μL of Recovery Agent to the sample and wait for 2 minutes. After the liquid layers separate, slowly remove 125 μL of the mixed liquid of Recovery Agent and Partitioning Oil (pink).

[0073] 1.3 Sample purification

[0074] 1) Take out Dynabeads MyOne SILANE (10X Genomics) from the 4 °C refrigerator, mix it well, and take 8 μL into a new 250 μL EPP tube. Then add 182 μL of Cleanup Buffer (10X Genomics), 5 μL of Reducing Agent B (10X Genomics), and 5 μL of Nuclease-free Water to the tube to prepare a total of 200 μL of Dynabeads Cleanup Mix.

[0075] 2) Add 200 μL of the prepared Dynabeads Cleanup Mix to the product of 1.2(3), mix well, let it stand at room temperature for 5 minutes, pipette and mix again, then let it stand at room temperature for another 5 minutes, and then place it on a magnetic rack.

[0076] 3) Use 300 μL of freshly prepared 80% ethanol, wait for 30 seconds in the tube, and discard the supernatant.

[0077] 4) Repeat the previous step once. Briefly centrifuge the centrifuge tube after removing the supernatant for 2 s, place it on the magnetic rack and let it stand until clear, and remove the remaining ethanol.

[0078] 5) Prepare Elution Solution I: Buffer EB 98 μL + 10% Tween 20 (freshly prepared) 1 μL + Reducing Agent B (10X Genomics) 1 μL, a total of 100 μL.

[0079] 6) After opening the lid and letting it dry for 1 minute, re-dissolve with 34.5 μL Elution Solution I, pipette to mix well, and place at room temperature (25°C) for 10 minutes.

[0080] 7) After 10 minutes, take out the 250 μL centrifuge tube, centrifuge for 2 seconds, place it on a magnetic rack, let it stand until it is clear, aspirate the supernatant, and collect it in another labeled 250 μL EP centrifuge tube.

[0081] 1.4 Primer digestion

[0082] 1) Add 1 μL each of Exonuclease I (E. coli, NEB) and RNase inhibitor (Invitrogen) to the purified product tube, then add 4 μL of NEB buffer 2 (10X, NEB) and place at 37°C for reaction for 30 min;

[0083] 2) Take out RNAClean XP Beads (Beckman CoμLter, Inc) 30 minutes in advance and place at room temperature. Vortex and mix thoroughly before use. Take 1.8 times the volume of RNAClean XP Beads and add it to the PCR product. Pipette gently for at least 10 times to mix thoroughly. Incubate at room temperature for 5 minutes.

[0084] 3) Place the centrifuge tube on the magnetic rack and let it stand for 2-5 minutes until the liquid becomes clear. Use a pipette to aspirate and discard the supernatant. Keep the centrifuge tube fixed on the magnetic rack, add 120 μL of freshly prepared 80% ethanol, let it stand at room temperature for 30 seconds, and then carefully discard the supernatant.

[0085] 4) Repeat the previous step once, discard the supernatant and place the tube on the magnetic rack to absorb the liquid at the bottom of the tube as much as possible;

[0086] Note: Do not pipette the beads as this will affect the yield.

[0087] 5) Keep the centrifuge tube fixed on the magnetic rack, open the centrifuge tube cover, and dry it at room temperature until the surface of the magnetic beads is not reflective or cracked;

[0088] 6) Remove the centrifuge tube from the magnetic stand, add 8.5 μL TE to elute the cDNA, mix well with a pipette and dissolve at room temperature for 5 minutes;

[0089] 7) Place the centrifuge tube on the magnetic stand and let it stand for 2 - 5 min until the liquid becomes clear. Transfer the supernatant to a new centrifuge tube.

[0090] 2. Template replacement

[0091] 2.1 cDNA synthesis (reverse transcription RT)

[0092] 1) Take the purified product (8 μl) of extended RNA for RT. The reaction system and reaction conditions are shown in Table 2 below:

[0093] Table 2

[0094]

[0095]

[0096] Among them, the RT primer sequence is 5’-CTACACGACGCTCTTCCGATCT-3’ (SEQ ID NO:4).

[0097] 2) After the reaction, purify the cDNA with 0.8X AMPure PB magnetic beads.

[0098] 3. PCR amplification

[0099] 1) Take 10 μL of the cDNA purified and recovered in the previous step to prepare the PCR system. The reaction system and reaction conditions are shown in Table 3 below:

[0100] Table 3

[0101]

[0102]

[0103] Among them, the PCR primer F sequence is 5’-CTACACGACGCTCTTCCGATCT-3’ (SEQ ID NO:1)

[0104] The PCR primer R sequence is 5’-AAGCAGTGGTATCAACGCAGAG-3’ (SEQ ID NO:2)

[0105] 2) After the reaction, transfer the PCR product to a 1.5 mL centrifuge tube and purify it with 0.8X AMPure PB magnetic beads.

[0106] 4. SMRTbell TM Library construction

[0107] 4.1 End repair and adapter ligation

[0108] 1) It is carried out using the NEBNext Ultra II DNA Library Prep Kit for Illumina. The end repair system is shown in the following table:

[0109] Table 4

[0110]

[0111] 2) After completing the end repair, adaptors are added. The reaction system is shown in the following table:

[0112] Table 5

[0113]

[0114] / 5Phos / NNNNNNNNNNNNNNNN(16bp barcode)

[0115] ATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATNNNNNNNNNNNNNNNN((16bp barcode)T(SEQ ID NO:3)

[0116] 4.2 Enzyme digestion reaction and magnetic bead purification

[0117] 1) Add the mixture shown in Table 6 to the product of the previous step. The detailed reaction system is shown in the following table:

[0118] Table 6

[0119]

[0120]

[0121] 2) After the reaction is completed, take a new 1.5 mL centrifuge tube, make a mark, and transfer all the reaction system to the corresponding new centrifuge tube.

[0122] 3) Add 0.5 times the volume (50 μL) of AMPure PB magnetic beads to the 1.5 mL centrifuge tube containing the enzyme digestion reaction product for purification, and finally redissolve it in 50 μL of elution buffer.

[0123] 4) Dilute 1 μL of the purified sample by 5 times, take 1 μL of the diluted sample for Qubit detection, and perform 2100 quality inspection on the remaining diluted sample.

[0124] 5. Sequencing on the machine

[0125] After library construction, the loading scheme provided by PacBio was adopted, and the preparation for sequencing was carried out according to the requirements of the Procedure&Checklist–Preparing Single-Cell IsoSeq TM Libraries Using ExpressTemplatePrep Kit 2.0 instruction manual.

[0126] 6. Test Results

[0127] The 4T1 cell line was used for library construction and sequencing of full-length transcripts containing poly(A).

[0128] 1) Library quality inspection results after leaving the library:

[0129] After library construction, the library was inspected by 2100. The inspection results showed that the main peaks of the library were all around 1.7k, which were all within the normal range. See Figure 2 .

[0130] Quality inspection results of the library for full-length transcripts containing poly(A)

[0131] 2) The length of poly(A) detected in the sequencing data obtained by the library construction method is shown in the following table and Figure 3 .

[0132] According to Figure 3 it can be known that: the length distribution of the polyA tails measured by the method of the present invention ranges from 30 nt to more than 400 nt, and the average polyA tail length is 83.21 nt.

[0133] Table 7

[0134] Library Average Poly(A) Length Full-length Transcriptome with Poly(A) 83.21nt SEQUENCE LISTING <110> BGI Tech Solutions (Shenzhen) Co., Ltd. <120> Method for amplifying or detecting full-length transcriptome cDNA containing full-length poly(A) <130> P20017311C <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer F <400> 1 ctacacgacg ctcttccgat ct 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer R <400> 2 aagcagtggt atcaacgcag ag 22 <210> 3 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Adapter sequence <220> <221> misc_feature <222> (46)..(61) <223> n is a, c, g, or t <400> 3 atctctctct tttcctcctc ctccgttgtt gttgttgaga gagatnnnnn nnnnnnnnnn 60 nt 62 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RT primer <400> 4 ctacacgacg ctcttccgat ct 22

Claims

1. A method for amplifying or detecting full-length transcript cDNA containing full-length poly(A) based on the 10X genomics single-cell platform, characterized in that, the method comprises: (1)Single-cell reaction preparation; it includes a) preparing a single-cell suspension; b) formulating a Master mix; c) mixing a) and b) to obtain a mixed solution and preparing droplets; wherein the Master mix includes dNTP, a DNA polymerase without 5'->3' exonuclease activity, and a buffer, and the buffer includes 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 and 1 mM DTT; (2) Terminal extension: Using 10×barcoded gel beads with Poly(dT) as a template, the transcript with poly(A) is extended at the terminal to obtain a single-stranded product 1; (3) Template replacement: Reverse transcribing the product 1, adding 3 bases C to the 3' end after the reverse transcription product; and adding a TSO primer for reaction to obtain product 2; (4) PCR amplification: Performing PCR amplification on the product 2; Before step (3), sample purification and primer digestion are performed on the product obtained in step (2); The primer digestion includes: 1) Adding an equal amount of Exonuclease I and RNase inhibitor into the purified product tube, then adding 10× NEB buffer2 and placing it at 37°C for reaction; 2) Taking 1.8 times the volume of room temperature RNAClean XP Beads and adding them to the PCR product, and mixing well, incubating at room temperature; 3) Standing for 2 - 5 min until the liquid becomes clear, discarding the supernatant; adding an equal volume of 80% ethanol, standing at room temperature and then discarding the supernatant; 4) Repeating step 3), discarding the supernatant and trying to suck dry the liquid at the bottom of the tube; 5) Drying at room temperature to make the surface of the magnetic beads have no reflection and no cracking; 6) Adding TE for cDNA elution, pipetting and mixing well and dissolving at room temperature; 7) Placing the centrifuge tube on a magnetic rack, standing until the liquid becomes clear, and transferring the supernatant to a new centrifuge tube.

2. The method according to claim 1, characterized in that, The single-cell reaction preparation described in step (1) includes: a) Preparing a single-cell suspension, and each 43.2 μL of the single-cell suspension is prepared by mixing 8 μL of RNase inhibitor and a standard cell with a concentration of 700 - 1200 cells / μL, and making up NF water; and / or, b) Formulating Master mix, and each 31.8 μL of the Master mix contains 6.1 μL of dNTP with a concentration of 10 μM, 9.7 μL of Klenow fragment with a concentration of 50 units / μL, 14 μL of 10×NEB buffer2, and 2 μL of reducing agent B; the buffer is 10×NEB buffer2; and / or, c) The preparation of droplets is as follows: adding the mixed solution, Gel Beads, and Partitioning Oil into rows labeled 1, 2, and 3 of GEMChip G in sequence, covering the GEM Chip G with a 10×Gasket and preparing droplets.

3. The method according to claim 2, characterized in that, The number of the standard cells is 10,000; and / or, In the droplet, the volume ratio of the single cell suspension, Master mix, Gel Beads and Partitioning Oil is 43.2:31.8:50:

45.

4. The method according to claim 1, It is characterized in that The terminal extension in step (2) includes: a) reacting the product obtained in step (1) at 37° C. for 1 hour; b) Adding Recovery Agent to the product obtained in step a) at a volume ratio of 4:5, and removing the mixed liquid of Recovery Agent and Partitioning Oil after the liquid is separated into layers.

5. The method according to claim 4, It is characterized in that 100 μL of the product obtained in a) was used, and 125 μL of the Recovery Agent described in b) was used.

6. The method according to claim 1, It is characterized in that Before step (3), the product obtained in step (2) is subjected to sample purification and primer digestion; The sample purification comprises: 1) Mix Dynabeads MyOne SILANE, Cleanup Buffer, Reducing Agent B and Nuclease-free Water to prepare Dynabeads Cleanup Mix; 2) mixing the Dynabeads Cleanup Mix and the product obtained in step (2) and letting it stand at room temperature; 3) Add 80% ethanol to Dynabeads Cleanup Mix in a volume ratio of 2:3 to 80% ethanol, let the tube stand, and discard the supernatant; 4) Add Elution Solution I to dissolve again; 5) Mix by pipetting and place at room temperature (25°C) for 10 min. And / or, the primer digestion comprises: 1) Add equal amounts of Exonuclease I and RNase inhibitor to the purified product tube, then add 10× NEB buffer 2 and place at 37°C for reaction; 2) Take 1.8 times the volume of room temperature RNAClean XP Beads and add it to the PCR product and mix thoroughly, then incubate at room temperature; 3) Let stand until the liquid is clear and discard the supernatant; add an equal volume of 80% ethanol, let stand at room temperature and discard the supernatant; 4) Repeat step 3), discard the supernatant and absorb the liquid at the bottom of the tube as much as possible; 5) Dry at room temperature to make the surface of the magnetic beads non-reflective and crack-free; 6) Add TE to elute cDNA, mix well with a pipette and dissolve at room temperature; 7) Place the centrifuge tube on a magnetic rack and let it stand until the liquid becomes clear. Transfer the supernatant to a new centrifuge tube.

7. The method according to claim 6, It is characterized in that During the sample purification: The volume ratio of the Dynabeads MyOne SILANE, the Cleanup Buffer, the Reducing Agent B, and Nuclease-free Water is 8:182:5:5, and the volume of the Dynabeads Cleanup Mix is 200 µL; and / or, 2) further includes pipetting and mixing again and standing at room temperature; the standing time is 5 minutes; and / or, 3) the standing time in it is 30 seconds; and / or, repeat step 3) once, centrifuge the centrifuge tube after discarding the supernatant for 2 s, stand until clear, and remove the residual ethanol; and / or, the volume ratio of the Elution Solution I to the mixed solution obtained in step 3) is 1:10 to 1:15; the Elution Solution I is prepared by mixing Buffer EB, 10% Tween 20, and Reducing Agent B, and the volume ratio of the Buffer EB, the 10% Tween 20, and the Reducing Agent B is 98:1:1; the Reducing Agent B is from 10X Genomics; and / or, in the primer digestion: 1) the reaction time in it is 30 min, and the volume ratio of the purified product, Exonuclease I, RNase inhibitor, and 10X NEB buffer 2 is (30 - 35):1:1:4; and / or, 2) the incubation time in it is 5 min; and / or, 3) stand for 2 - 5 min until the liquid is clear, discard the supernatant; add an equal volume of 80% ethanol, stand at room temperature for 30 seconds, and then discard the supernatant; and / or, 6) the dissolution time in it is 5 min; and / or, 7) the time for the liquid to become clear in it is 2 - 5 min.

8. The method according to any one of claims 1 to 7, characterized in that in step (3), superscriptTMII / IV is used for the reverse transcription; and / or, in step (4), each 50 μL of the PCR amplification system includes: 9 μL of the product obtained in step (3), 25 μL of 2X KAPA HiFi HotStart ReadyMix, 2 μL of the upstream primer with a concentration of 10 µM as shown in SEQ ID NO:1, 2 μL of the downstream primer with a concentration of 10 µM as shown in SEQ ID NO:2, and 12 μL of NF water.

9. A method for constructing a library of a full-length transcriptome containing poly(A), characterized in that the library construction method includes the method according to any one of claims 1 to 8.

10. The library construction method according to claim 9, characterized in that it further includes: performing end repair on the product obtained in step (4).

11. The library construction method according to claim 10, characterized in that The volume of the reaction system for end repair is 30 μL.

12. The library construction method according to claim 11, wherein, each 30 μL of the reaction system contains: 25 μL of the product, 3.5 μL of NEBNext Ultra II End Prep Reaction Buffer, and 1.5 μL of NEBNext Ultra II End Prep Enzyme Mix.

13. The library construction method according to claim 10, wherein, the reaction conditions for end repair are as follows: reacting the reaction system at 20°C for 30 min and then at 65°C for 30 min.

14. The library construction method according to any one of claims 9 to 13, wherein, an adaptor is added after the end repair, and the sequence of the adaptor is as shown in SEQ ID NO:

3.

15. The library construction method according to claim 14, wherein, an enzymatic digestion reaction and magnetic bead purification are carried out after adding the adaptor.

16. The library construction method according to claim 15, wherein, in the reaction system for enzymatic digestion, each 95 μL contains 42.5 μL of the product after adding the adaptor, 10 μL of reaction buffer, 1 μL of exonuclease I with a concentration of 20 U / μL, 1 μL of exonuclease III with a concentration of 100 U / μL, and 40.5 μL of nuclease-free water.

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