Single cell mRNA and sRNA co-sequencing method

Abstract

The invention provides a single-cell mRNA and sRNA co-sequencing method, which comprises the following steps: preparing a single-cell suspension, adding the single-cell suspension into a micropore of a chip of a single-cell operation system, and selecting a micropore of a single living cell to carry out an experiment, and then carrying out cell lysis reaction, sRNA 3'end ligation reaction, mRNA reverse transcription reaction, sRNA free linker removal reaction, sRNA 5 'end ligation reaction, sRNA reverse transcription reaction and first PCR (Polymerase Chain Reaction) reaction, and screening and recovering products and carrying out second PCR reaction, thereby obtaining the single-cell mRNA and sRNA co-sequencing library which can be directly used for on-machine sequencing. The method has the advantages of high accuracy, high sensitivity, good repeatability and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of single-cell sequencing, and relates to a method for co-sequencing single-cell message RNA (mRNA) and small RNA (sRNA). BACKGROUND

[0002] miRNA is a non-coding RNA widely present in eukaryotes that can regulate the expression of other genes. miRNA is an indispensable regulator of many biological pathways and diseases, and is partially complementary to one or more mRNA molecules, which can accelerate the degradation of target mRNA or inhibit the translation of target mRNA through translation inhibition, mRNA cleavage and de-adenylation, and is involved in post-transcriptional regulation of mRNA. miRNA plays an important role in regulating gene expression, cell cycle, and biological development timing. Therefore, double-genomic analysis of mRNA and miRNA will help to understand the functional mechanism of miRNA. miRNA is a kind of sRNA with important biological regulation function, which participates in various biological processes by complementary pairing with target RNA.

[0003] In the prior art, early miRNA and mRNA co-sequencing adopts a simple method of "lysis of cells and separate library construction". For example, the article "Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation" (Non-patent document 1) published by Nayi Wang et al. In this method, as shown in Figure 1 , the lysis cell contents are divided into two equal parts and then subjected to mRNA and sRNA library construction sequencing, or mRNA contents are extracted using polyadenylated enrichment beads for mRNA sequencing, and the remaining RNA contents are used for sRNA sequencing. However, this method is very inefficient and complicated to operate, and is difficult to use for large-scale applications (such as analysis of complex biopsy samples).

[0004] The recently reported Smart-seq-total method (for example, the article "Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states" published by Alina Isakova et al., hereinafter referred to as Non-patent document 2). As shown in Figure 2As shown, coding and non-coding genes (including miRNA) can be captured simultaneously in the same reaction tube, but the content of miRNA in this analysis is very low. In addition, the operation of the DASH (Depletion of Abundant Sequences by Hybridization) method to remove rRNA fragments is complicated, the library construction efficiency is low, and it is difficult to be widely used.

[0005] Therefore, although the high-throughput analysis method of single-cell mRNA-seq has been successfully applied to many research fields of biology and medicine. There is no efficient method for co-expression analysis of single-cell miRNA and mRNA. SUMMARY

[0006] Based on the defects existing in the prior art, the purpose of the present application is to provide an efficient method for co-sequencing single-cell mRNA and sRNA.

[0007] The purpose of the present application is achieved by the following technical means:

[0008] In one aspect, the present application provides a method for co-sequencing single-cell mRNA and sRNA, comprising the following steps:

[0009] (1) preparing a single-cell suspension, adding it to the microwells of the chip of the single-cell operation system, and selecting the microwells of single living cells for experiment;

[0010] (2) cell lysis reaction;

[0011] (3) sRNA 3' end ligation reaction;

[0012] (4) mRNA reverse transcription reaction;

[0013] (5) sRNA free adapter removal reaction;

[0014] (6) sRNA 5' end ligation reaction;

[0015] (7) sRNA reverse transcription reaction;

[0016] (8) first PCR reaction, purifying and recovering the PCR-1 product, dividing the PCR-1 product into two parts, and screening and recovering mRNA and sRNA libraries, respectively;

[0017] (9) second PCR reaction, which is performed by taking the screened and recovered PCR-1 product as a template, and then purifying and recovering the PCR-2 product, thereby obtaining a single-cell mRNA and sRNA co-sequencing library that can be directly used for sequencing, and performing sequencing.

[0018] In the sRNA 3' end ligation reaction of step (3), the nucleotide sequence of the 3' adaptor used is shown in SEQ ID NO: 1.

[0019] In the sRNA 5' end ligation reaction of step (6), the nucleotide sequence of the 5' adaptor used is shown in SEQ ID NO: 2.

[0020] The single-cell mRNA and sRNA co-sequencing process of the present application modifies and improves the 3' adaptor and the 5' adaptor, and the nucleotide sequence of the 3' adaptor is as follows: (5'→3')

[0021] SEQ ID NO: 1: rApp / 5' - GTCGACTGATACATCGGTCTGTCGACTG - 3' NN CTGTAGGCACCATCAAT / ddC

[0022] (rApp represents acylated adenosine; ddC represents double deoxy cytosine; N represents A, G, C or T, which is a random deoxyribonucleic acid)

[0023] The nucleotide sequence of the 5' adaptor is as follows: (5'→3')

[0024] 5' biotin / rGrArCrUrGrArCrUrUrGrUrCrArCrGrUrUrGrNrNrUrUrGrNrNrUrUrGrNrN (SEQ ID NO: 2)

[0025] (rN represents rA, rC, rG or rU, which is a random ribonucleic acid)

[0026] In the above method, the process of preparing the single-cell suspension further includes the steps of staining and counting the cells. The cells can include A549 cells, mouse lung tissue single cells, and the like. The tissue or single-cell suspension can be fixed with dithiobis(succinimidyl propionate) and then stored at 4°C for up to 72 hours or at -80°C for up to three months.

[0027] In the above method, step (2) can be transferring the chip to a -80°C refrigerator for freezing for 1 hour, incubating at 25°C for 5 minutes, and then to room temperature.

[0028] In the above method, step (3) is adding the sRNA 3' end ligation reaction mixture to the selected microwells, then transferring the chip to a PCR instrument, and performing the sRNA 3' end ligation reaction program; preferably, the components of the sRNA 3' end ligation reaction mixture include a 3' adaptor, T4 RNA ligase 2 (truncated KQ), T4 RNA ligation buffer, and a ribonuclease inhibitor. The sRNA 3' end ligation reaction mixture can also include a Triton x-100 lysis solution and a ribonuclease inhibitor.

[0029] In the above method, preferably, the sRNA 3’ end ligation reaction procedure is 6 hours of incubation at 25°C, followed by 8-10 hours of reaction at 4°C.

[0030] Preferably, prior to performing the sRNA 3’ end ligation reaction procedure, the chip can also be heated at 75°C for 5 minutes.

[0031] In the above method, the mRNA reverse transcription reaction of step (4) is that the mRNA reverse transcription mixture is added to the selected microwells, then the reverse transcription reaction solution is added thereto, and then the chip is moved to a PCR instrument to perform a reverse transcription reaction procedure; preferably, the mRNA reverse transcription mixture comprises mRNA reverse transcription primers and reverse transcription buffer. Further preferably, the components of the mRNA reverse transcription mixture comprise 168 μl of 2 uM mRNA reverse transcription primers and 168 μl of mRNA reverse transcription reaction solution (dNTP, reverse transcriptase, strand displacement primer, ribonuclease inhibitor). Further preferably, the reverse transcriptase is Maxima H Minus Reverse Transcriptase (EP0753 Thermo Fisher). Other reverse transcriptases that can be used for strand displacement reactions include SuperScript TM II Reverse Transcriptase (18064071, Thermo Fisher), SuperScript TM IV Reverse Transcriptase (18091050, Thermo Fisher), SMARTScribe TM Reverse Transcriptase (639538, Clontech), Maxima Reverse Transcriptase (EP0743, Thermo Fisher) can also be used as an alternative to Maxima H Minus Reverse Transcriptase.

[0032] In the above method, the sequence of the mRNA reverse transcription primer is as shown in SEQ ID NO: 3, wherein the reverse transcription primer is longer than a conventional reverse transcription primer. The sequence of the strand displacement primer is as shown in SEQ ID NO: 4.

[0033] In the above method, the nucleotide sequence of the mRNA reverse transcription primer is as follows: (5’→3’)

[0034] 5’ biotin / GATTGATGGTGCCTACAGDDNDDNDDNDDTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 3)

[0035] In the above method, the nucleotide sequence of the strand displacement primer is as follows: (5’→3’)

[0036] 5' biotin / GACTGACTTGTCACGTTT / rG / / rG / / +G / (SEQ ID NO: 4)

[0037] (rG represents ribonucleic acid G, and +G represents a deoxyribonucleic acid G with a locked nucleotide modification)

[0038] In the above method, preferably, the reverse transcription reaction procedure is 90 minutes of reaction at 50°C, followed by 5 minutes of heating at 85°C.

[0039] In the above method, preferably, the free linker removal reaction of sRNA in step (5) is to add the linker removal reaction solution to the selected microwells, and then move the chip to a PCR instrument to perform a linker removal reaction procedure; preferably, the components of the linker removal reaction solution include exonuclease, 5' deadenylase, and ribonuclease inhibitor.

[0040] In the above method, preferably, the linker removal reaction procedure is 30 minutes of incubation at 30°C, followed by 60 minutes of incubation at 37°C, and finally 10 minutes of heating at 75°C.

[0041] In the above method, preferably, before the free linker removal reaction, it further includes adding the sRNA reverse transcription mixture to the selected microwells, and then moving the chip to a PCR instrument to perform a heating reaction; preferably, the components of the sRNA reverse transcription mixture include a labeled sRNA reverse transcription primer and an exonuclease buffer. The labeled sRNA reverse transcription primer includes one or more of SEQ ID NO: 5 to SEQ ID NO: 76.

[0042] The nucleotide sequence of the labeled sRNA reverse transcription primer is specifically as follows: (5'→3')

[0043] CTTGGCACCCGAGAATTCCAGATCGCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 5)

[0044] CTTGGCACCCGAGAATTCCAGCAGGANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 6)

[0045] CTTGGCACCCGAGAATTCCAGTCACTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 7)

[0046] CTTGGCACCCGAGAATTCCATCCTGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 8)

[0047] CTTGGCACCCGAGAATTCCATTGAGGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 9)

[0048] CTTGGCACCCGAGAATTCCAAACCACNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 10)

[0049] CTTGGCACCCGAGAATTCCAACTAGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 11)

[0050] CTTGGCACCCGAGAATTCCAAATGGANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 12)

[0051] CTTGGCACCCGAGAATTCCAACTTCGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 13)

[0052] CTTGGCACCCGAGAATTCCAAGCGTTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 14)

[0053] CTTGGCACCCGAGAATTCCAATACCANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 15)

[0054] CTTGGCACCCGAGAATTCCACAGTTCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 16)

[0055] CTTGGCACCCGAGAATTCCACGAAGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 17)

[0056] CTTGGCACCCGAGAATTCCACGTGAGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 18)

[0057] CTTGGCACCCGAGAATTCCACTCCTGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 19)

[0058] CTTGGCACCCGAGAATTCCAGAACTTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 20)

[0059] CTTGGCACCCGAGAATTCCAGACTGGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 21)

[0060] CTTGGCACCCGAGAATTCCAGCATACNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 22)

[0061] CTTGGCACCCGAGAATTCCATCAATGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 23)

[0062] CTTGGCACCCGAGAATTCCATGAGCCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 24)

[0063] CTTGGCACCCGAGAATTCCATGGCATNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 25)

[0064] CTTGGCACCCGAGAATTCCAAATCTGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 26)

[0065] CTTGGCACCCGAGAATTCCAAAGACTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 27)

[0066] CTTGGCACCCGAGAATTCCAAGCTGANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 28)

[0067] CTTGGCACCCGAGAATTCCAATAGACNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 29)

[0068] CTTGGCACCCGAGAATTCCACCACATNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 30)

[0069] CTTGGCACCCGAGAATTCCACGAGTANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 31)

[0070] CTTGGCACCCGAGAATTCCACTAACGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 32)

[0071] CTTGGCACCCGAGAATTCCACTCGGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 33)

[0072] CTTGGCACCCGAGAATTCCAGAGAACNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 34)

[0073] CTTGGCACCCGAGAATTCCAGTGCGANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 35)

[0074] CTTGGCACCCGAGAATTCCATACGCANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 36)

[0075] CTTGGCACCCGAGAATTCCATCGTAGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 37)

[0076] CTTGGCACCCGAGAATTCCATGTTCTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 38)

[0077] CTTGGCACCCGAGAATTCCAAGGATGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 39)

[0078] CTTGGCACCCGAGAATTCCAATCAGCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 40)

[0079] CTTGGCACCCGAGAATTCCACCGTCTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 41)

[0080] CTTGGCACCCGAGAATTCCACTTCACNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 42)

[0081] CTTGGCACCCGAGAATTCCAGAAGAGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 43)

[0082] CTTGGCACCCGAGAATTCCAGGAACANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 44)

[0083] CTTGGCACCCGAGAATTCCAGGCTTCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 45)

[0084] CTTGGCACCCGAGAATTCCAGGTGGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 46)

[0085] CTTGGCACCCGAGAATTCCATCACGCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 47)

[0086] CTTGGCACCCGAGAATTCCAACTCACNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 48)

[0087] CTTGGCACCCGAGAATTCCAAGAGATNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 49)

[0088] CTTGGCACCCGAGAATTCCAAGGACANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 50)

[0089] CTTGGCACCCGAGAATTCCAATCCGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 51)

[0090] CTTGGCACCCGAGAATTCCAATGTTGNNNNNNGATTGATGGTGCCTACAG(SEQ ID NO:52)

[0091] CTTGGCACCCGAGAATTCCACACGACNNNNNNGATTGGTGCCTACAG(SEQ ID NO:53)

[0092] CTTGGCACCCGAGAATTCCACAGATTNNNNNNGATTGGTGCCTACAG(SEQ ID NO:54)

[0093] CTTGGCACCCGAGAATTCCAGATGTANNNNNNGATTGGTGCCTACAG(SEQ ID NO:55)

[0094] CTTGGCACCCGAGAATTCCAGCACCTNNNNNNGATTGGTGCCTACAG(SEQ ID NO:56)

[0095] CTTGGCACCCGAGAATTCCAGCCATGNNNNNNGATTGGTGCCTACAG(SEQ ID NO:57)

[0096] CTTGGCACCCGAGAATTCCAGGCTAANNNNNNGATGATGGTGCCTACAG(SEQ ID NO:58)

[0097] CTTGGCACCCGAGAATTCATAGCGANNNNNNGATTGATGGTGCCTACAG(SEQ ID NO:59)

[0098] CTTGGCACCCGAGAATTCCATCATTCNNNNNNGATTGGTGCCTACAG(SEQ ID NO:60)

[0099] CTTGGCACCCGAGAATTCCATTGGCTNNNNNNGATTGGTGCCTACAG(SEQ ID NO:61)

[0100] CTTGGCACCCGAGAATTCCAAAGGAGNNNNNNGATTGGTGCCTACAG(SEQ ID NO:62)

[0101] CTTGGCACCCGAGAATTCCACATCCTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 64)

[0102] CTTGGCACCCGAGAATTCCACGACAANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 65)

[0103] CTTGGCACCCGAGAATTCCACTAATCNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 66)

[0104] CTTGGCACCCGAGAATTCCACTCTATNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 67)

[0105] CTTGGCACCCGAGAATTCCAGACACANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 68)

[0106] CTTGGCACCCGAGAATTCCAGGATTGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 69)

[0107] CTTGGCACCCGAGAATTCCATAAGGTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 70)

[0108] CTTGGCACCCGAGAATTCCAAACAGGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 71)

[0109] CTTGGCACCCGAGAATTCCAACAGTGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 72)

[0110] CTTGGCACCCGAGAATTCCAAGTTAGNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 73)

[0111] CTTGGCACCCGAGAATTCCAGTTGTTNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 74)

[0112] CTTGGCACCCGAGAATTCCAATGAATNNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 74)

[0113] CTTGGCACCCGAGAATTCCACCAAGANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 75)

[0114] CTTGGCACCCGAGAATTCCAGAGTCANNNNNNGATTGATGGTGCCTACAG (SEQ ID NO: 76)

[0115] In the method described above, preferably, the temperature for the heating reaction is 70°C, and the reaction time is 2 minutes.

[0116] In the method described above, the sRNA 5' end ligation reaction of step (6) is to add the sRNA 5' end ligation reaction solution into the selected microwells, and then move the chip into the PCR instrument to execute the sRNA 5' end ligation reaction program; preferably, the components of the sRNA 5' end ligation reaction solution include 5' adapter, T4 RNA ligase 1, T4 RNA ligation buffer, ATP, and ribonuclease inhibitor.

[0117] In the method described above, preferably, the program for the sRNA 5' end ligation reaction is 25°C incubation for 2 hours, and then 65°C heating for 15 minutes.

[0118] In the method described above, the sRNA reverse transcription reaction of step (7) is to add the sRNA reverse transcription reaction solution into the selected microwells, and then move the chip into the PCR instrument to execute the sRNA reverse transcription reaction program; preferably, the components of the sRNA reverse transcription reaction solution include single-strand synthesis buffer, DTT, dNTP, ribonuclease inhibitor, and reverse transcriptase.

[0119] Preferably, the sRNA reverse transcription reaction program is 52°C reaction for 50 minutes, and then 70°C heating for 15 minutes.

[0120] In the method described above, the first PCR reaction of step (8) is to add the PCR-1 reaction solution into the selected microwells, and then move the chip into the PCR instrument to execute the PCR-1 reaction program; preferably, the components of the PCR-1 reaction solution include labeled PCR-1 primer, dNTP, PCR buffer, and DNA polymerase.

[0121] The sequence of the labeled PCR-1 primer includes one or more of SEQ ID NO: 77 to SEQ ID NO: 148.

[0122] The nucleotide sequence of the labeled PCR-1 primer is as follows: (5'→ 3')

[0123] GTTCAGAGTTCTACAGTCCGACGATCAACCAAGACTGACTTGTCACGTT (SEQ ID NO: 77)

[0124] GTTCAGAGTTCTACAGTCCGACGATCCGATAGGACTGACTTGTCACGTT (SEQ ID NO: 78)

[0125] GTTCAGAGTTCTACAGTCCGACGATCAGAAGAGACTGACTTGTCACGTT (SEQ ID NO: 79)

[0126] GTTCAGAGTTCTACAGTCCGACGATCGAGCCTGACTGACTTGTCACGTT (SEQ ID NO: 80)

[0127] GTTCAGAGTTCTACAGTCCGACGATCTAGTCAGACTGACTTGTCACGTT (SEQ ID NO: 81)

[0128] GTTCAGAGTTCTACAGTCCGACGATCACTGCAGACTGACTTGTCACGTT (SEQ ID NO: 82)

[0129] GTTCAGAGTTCTACAGTCCGACGATCCAGCATGACTGACTTGTCACGTT (SEQ ID NO: 83)

[0130] GTTCAGAGTTCTACAGTCCGACGATCCCGCCTGACTGACTTGTCACGTT (SEQ ID NO: 84)

[0131] GTTCAGAGTTCTACAGTCCGACGATCCCTAGCTGACTGACTTGTCACGTT (SEQ ID NO: 85)

[0132] GTTCAGAGTTCTACAGTCCGACGATCCGCAACTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0133] GTTTCTAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0134] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0135] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0136] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0137] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0138] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0139] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0140] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0141] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0142] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0143] GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 85) GTTCAGAGTTCTACAGTCCGACGATCGCGGTTTGACTGACTTGTCACGTT (SEQ ID NO: 86)

[0144] GTTTCTAGAGTTCTACAGTCCGACGATCGAGTGGGTGACTGACTTGTCACGTT (SEQ ID NO: 97)

[0145] GTTTCTAGAGTTCTACAGTCCGACGATCGGTAGCGTGACTGACTTGTCACGTT (SEQ ID NO: 98)

[0146] GTTTCTAGAGTTCTACAGTCCGACGATCATGAGCAGTGACTGACTTGTCACGTT (SEQ ID NO: 101)

[0147] GTTTCTAGAGTTCTACAGTCCGACGATCATTCCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 102)

[0148] GTTTCTAGAGTTCTACAGTCCGACGATCCAAAAGAGTGACTGACTTGTCACGTT (SEQ ID NO: 103)

[0149] GTTTCTAGAGTTCTACAGTCCGACGATCCAACTAAGTGACTGACTTGTCACGTT (SEQ ID NO: 104)

[0150] GTTTCTAGAGTTCTACAGTCCGACGATCCACCGGAGTGACTGACTTGTCACGTT (SEQ ID NO: 105)

[0151] GTTTCTAGAGTTCTACAGTCCGACGATCCACGATAGTGACTGACTTGTCACGTT (SEQ ID NO: 106)

[0152] GTTTCTAGAGTTCTACAGTCCGACGATCCACTCAAGTGACTGACTTGTCACGTT (SEQ ID NO: 107)

[0153] GTTTCTAGAGTTCTACAGTCCGACGATCCACGATAGTGACTGACTTGTCACGTT (SEQ ID NO: 108)

[0154] GTTTCTAGAGTTCTACAGTCCGACGATCCACTCAAGTGACTGACTTGTCACGTT (SEQ ID NO: 109)

[0155] GTTCAGAGTTCTACAGTCCGACGATCCAGGCGTAGTGACTGACTTGTCACGTT (SEQ ID NO: 109)

[0156] GTTCAGAGTTCTACAGTCCGACGATCCATGGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 110)

[0157] GTTCAGAGTTCTACAGTCCGACGATCCATTTTTAGTGACTGACTTGTCACGTT (SEQ ID NO: 111)

[0158] GTTCAGAGTTCTACAGTCCGACGATCCCAACATAGTGACTGACTTGTCACGTT (SEQ ID NO: 112)

[0159] GTTCAGAGTTCTACAGTCCGACGATCCGGAATTAGTGACTGACTTGTCACGTT (SEQ ID NO: 113)

[0160] GTTCAGAGTTCTACAGTCCGACGATCCTAGCTTAGTGACTGACTTGTCACGTT (SEQ ID NO: 114)

[0161] GTTCAGAGTTCTACAGTCCGACGATCCTATACTAGTGACTGACTTGTCACGTT (SEQ ID NO: 115)

[0162] GTTCAGAGTTCTACAGTCCGACGATCCTCAGATAGTGACTGACTTGTCACGTT (SEQ ID NO: 116)

[0163] GTTCAGAGTTCTACAGTCCGACGATCGACGACCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 117)

[0164] GTTCAGAGTTCTACAGTCCGACGATCTAATCGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 118)

[0165] GTTCAGAGTTCTACAGTCCGACGATCTACAGCCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 119)

[0166] GTTCAGAGTTCTACAGTCCGACGATCTATAATCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 120)

[0167] GTTCAGAGTTCTACAGTCCGACGATCTCATTCCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 121)

[0168] GTTCAGAGTTCTACAGTCCGACGATCTCCCGACTAGTGACTGACTTGTCACGTT (SEQ ID NO: 122)

[0169] GTTCAGAGTTCTACAGTCCGACGATCTCGAAGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 123)

[0170] GTTCAGAGTTCTACAGTCCGACGATCTCGGCACTAGTGACTGACTTGTCACGTT (SEQ ID NO: 124)

[0171] GTTCAGAGTTCTACAGTCCGACGATCGGAGTTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 125)

[0172] GTTCAGAGTTCTACAGTCCGACGATCCCTTCAGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 126)

[0173] GTTCAGAGTTCTACAGTCCGACGATCCGAATAGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 127)

[0174] GTTCAGAGTTCTACAGTCCGACGATCCGGAGAGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0175] GTTTCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 127) GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0176] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0177] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0178] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0179] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0180] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0181] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0182] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0183] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0184] GTTCCTAGAGTTCTACAGTCCGACGATCGAAGCTGCTAGTGACTGACTTGTCACGTT (SEQ ID NO: 128)

[0185] GTTTCTAGAGTTCTACAGTCCGACGATCTTACTTAGCTAGTGACTGACTTGTCA CGTT (SEQ ID NO: 137)

[0186] GTTTCTAGAGTTCTACAGTCCGACGATCTTCTGACTAGCTAGGACTGACTTGTCA CGTT (SEQ ID NO: 138)

[0187] GTTTCTAGAGTTCTACAGTCCGACGATCTGGTCCCTAGCTAGGACTGACTTGTCA CGTT (SEQ ID NO: 139)

[0188] GTTTCTAGAGTTCTACAGTCCGACGATCTTACTTAGCTAGTGACTGACTTGTCA CGTT (SEQ ID NO: 140)

[0189] GTTTCTAGAGTTCTACAGTCCGACGATCTTCTGACTAGCTAGGACTGACTTGTCA CGTT (SEQ ID NO: 141)

[0190] GTTTCTAGAGTTCTACAGTCCGACGATCTGGTCCCTAGCTAGGACTGACTTGTCA CGTT (SEQ ID NO: 142)

[0191] GTTTCTAGAGTTCTACAGTCCGACGATCTTACTTAGCTAGTGACTGACTTGTCA CGTT (SEQ ID NO: 143)

[0192] GTTTCTAGAGTTCTACAGTCCGACGATCTTCTGACTAGCTAGGACTGACTTGTCA CGTT (SEQ ID NO: 144)

[0193] GTTTCTAGAGTTCTACAGTCCGACGATCTGGTCCCTAGCTAGGACTGACTTGTCA CGTT (SEQ ID NO: 145)

[0194] GTTTCTAGAGTTCTACAGTCCGACGATCTTACTTAGCTAGTGACTGACTTGTCA CGTT (SEQ ID NO: 146)

[0195] In the method, preferably, the PCR-1 reaction procedure is 95℃ for 3 minutes, then 12-14 cycles of 95℃ for 30 seconds, then 65℃ for 30 seconds, then 72℃ for 1 minute, and finally 72℃ for 5 minutes.

[0196] In the method, step (8) purifies the PCR-1 product by using nucleic acid purification magnetic beads (1.8x), and then performs fragment screening and recovery on the PCR-1 product; preferably, the product fragment size distribution is detected by using an Agilent 2100 bioanalyzer, then quantified by using a Qubit dsDNA HS assay kit, and the product fragment is screened by using a Pippin Prep; preferably, the sRNA library is recovered in a range of 134-162bp, and the mRNA library is recovered in a range of 270-650bp.

[0197] In the method, the second PCR reaction is performed by using the recovered PCR-1 product as a template, preparing a PCR-2 reaction solution, and performing a PCR-2 reaction procedure; preferably, the components of the PCR-2 reaction solution include SCSR-PCR-1 primers, labeled SCSR-PCR-2 primers, dNTPs, a PCR buffer, and a DNA polymerase.

[0198] In the method, the sequence of the SCSR-PCR-1 primer is shown in SEQ ID NO: 149, and the sequence of the labeled SCSR-PCR-2 primer includes one or more of SEQ ID NO: 150-SEQ ID NO: 161.

[0199] The nucleotide sequence of the SCSR-PCR-1 primer is specifically as follows: (5’→3’)

[0200] AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGAC GA (SEQ ID NO: 149)

[0201] The nucleotide sequence of the labeled SCSR-PCR-2 primer is specifically as follows: (5’→3’)

[0202] CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 150)

[0203] CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 151)

[0204] CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 152)

[0205] CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 153)

[0206] CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 154)

[0207] CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 155)

[0208] CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 156)

[0209] CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 157)

[0210] CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 158)

[0211] CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 159)

[0212] CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 160)

[0213] CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCCTTGGCA CCCGAGAATTCCA (SEQ ID NO: 161)

[0214] In the method, the PCR-2 reaction procedure is 95°C for 3 minutes, then 6-12 cycles of 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute, and finally 72°C for 5 minutes.

[0215] Preferably, the mRNA PCR-2 product is purified by using nucleic acid purification magnetic beads (1.6x).

[0216] In the method, preferably, the content of the library is determined by using a Qubit high-sensitivity kit, the size distribution is determined by using an Agilent 2100 bioanalyzer, and the quality is detected by using a library quantification kit.

[0217] In the method, preferably, the single-cell operation system is ICELL8.

[0218] Preferably, the cells include A549 cells and mouse lung tissue single cells.

[0219] In another aspect, the present application also provides an mRNA and sRNA co-sequencing library constructed by the above method.

[0220] Advantages

[0221] The present application introduces mRNA analysis (PSCSR-seqV2) on the basis of parallel single-cell sRNA sequencing technology (PSCSR-seq). The present application shows that PSCSR-seqV2 can overcome the limitations of existing methods, can simultaneously analyze mRNA and miRNA in a complete single cell, can detect twice as many miRNA species as other similar methods under the same sequencing depth of a single cell, and has the sensitivity of mRNA matching other methods. The present application innovatively designs an experimental method for obtaining sRNA and mRNA library information on a chip containing 5184 microwells, making high-throughput sequencing possible. PSCSR-seqV2 is expected to be widely used in cancer research, treatment application of mRNA optimization, and miRNA and mRNA expression profile analysis in life sciences. BRIEF DESCRIPTION OF DRAWINGS

[0222] Figure 1 is a schematic illustration of the early miRNA and mRNA co-sequencing technology of Non-Patent Literature 1. It can include the following steps:

[0223] Cell lysis - aliquot into two - one for sRNA library, i.e. 3' adapter ligation - free adapter removal - 5' adapter ligation - reverse transcription - first PCR amplification - second PCR amplification - PAGE gel recovery - sequencing; the other half for mRNA library, i.e. strand displacement reverse transcription - first PCR amplification - enzymatic fragmentation of PCR products - second PCR amplification - sequencing.

[0224] Figure 2 is a schematic illustration of the Smart-seq-total method of non-patent document 2. It can include the following steps:

[0225] Cell lysis - total RNA 3' end polyadenylated - strand displacement reverse transcription - enzymatic removal of strand displacement primer - first PCR (cDNA amplification) - second PCR (direct addition of index tag or addition of tag after enzymatic fragmentation) - removal of rRNA fragments using DASH method - pippin prep gel recovery - sequencing.

[0226] Figure 3 is a schematic illustration of the parallel single-cell mRNA and sRNA co-sequencing technology (PSCSR-seq V2) of the present application.

[0227] Figure 4 is a result graph showing the detection of mRNA species and miRNA species using different reverse transcriptases in the reverse transcription step.

[0228] Figure 5 is a comparison graph of mRNA species and miRNA species measured using the co-sequencing technology of the present application and the co-sequencing technology of non-patent document 1.

[0229] Figure 6 is a graph showing the effect of reverse transcription primers of different lengths (the number of T is selected to be 40 or more, such as 50T) on byproducts and gene mapping rate.

[0230] Figure 7 is a graph showing the effect of the reverse transcription primers (the number of T is more than 40) of the present application and 40T on gene mapping rate and gene number detection. DETAILED DESCRIPTION

[0231] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it cannot be understood as limiting the scope of implementation of the present application.

[0232] The following combines Figure 3 The parallel single-cell mRNA and sRNA co-sequencing technology (PSCSR-seq V2) of the present application is described.

[0233] The PSCSR-seq V2 of the present application can comprise the following steps:

[0234] (1) Stain and count cells, and prepare a single cell suspension. The tissue or single cell suspension can be fixed with dithiobis(succinimidyl propionate) and stored at 4°C for up to 72 hours or at -80°C for up to three months.

[0235] (2) Add cells to 5184 microwells of an icell8 350v chip using the MSND system, take a photo of each microwell using an imaging system, and select a single live cell using software.

[0236] (3) Transfer the chip to a -80°C freezer for 1 hour of storage, and incubate at 25°C for 5 minutes to room temperature.

[0237] (4) Heat the sRNA 3' end adapter at 70°C for 2 minutes, and prepare a 3' end ligation reaction solution. Add the 3' end ligation reaction solution to the selected microwells using the MSND system, and transfer the chip to a PCR instrument to perform an sRNA 3' end ligation reaction program.

[0238] (5) Add mRNA reverse transcription primers to the selected microwells using the MSND system, and transfer the chip to a PCR instrument for heating at 95°C for 3 minutes.

[0239] (6) Add mRNA reverse transcription reaction solution to the selected microwells using the MSND system, and transfer the chip to a PCR instrument to perform an mRNA reverse transcription program.

[0240] (7) Add sRNA reverse transcription primers with tags to the selected microwells using the MSND system, and transfer the chip to a PCR instrument for heating at 70°C for 2 minutes.

[0241] (8) Add a linker removal reaction solution to the selected microwells using the MSND system, and transfer the chip to a PCR instrument to perform a 3' linker removal reaction program.

[0242] (9) Heat the sRNA 5' end adapter at 70°C for 2 minutes, and prepare a 5' end ligation reaction solution. Add the 5' end ligation reaction solution to the selected microwells using the MSND system, and transfer the chip to a PCR instrument to perform an sRNA 5' end ligation reaction program.

[0243] (10) Add sRNA reverse transcription reaction solution to the selected microwells using the MSND system, and transfer the chip to a PCR instrument to perform an sRNA reverse transcription reaction program.

[0244] (11) PCR-1 primers with tags are added to the corresponding positions in the 384-well plate, and PCR reaction solution is added to each well. The PCR reaction solution is added to the selected microwells using the MSND system, and the chip is moved to the PCR instrument to perform the PCR-1 reaction program.

[0245] (12) The reaction solution in the chip is collected into a collection tube, and the PCR-1 product is recovered by magnetic bead purification.

[0246] (13) The PCR-1 product is aliquoted into two parts, one of which is subjected to sRNA library fragment screening and recovery on a pippin, and the other is subjected to mRNA library fragment screening and recovery on a pippin.

[0247] (14) PCR-2 reaction solution is prepared, and the screened PCR-1 product is used as a template to perform the PCR-2 reaction program on a PCR instrument.

[0248] (15) The sRNA and mRNA library PCR-2 products are recovered by nucleic acid purification magnetic beads.

[0249] (16) Quantitative analysis is performed using qubit 2.0, fragment size analysis is performed using Agilent 2100, absolute quantification is performed using QPCR, and sequencing is performed on a machine.

[0250] Example

[0251] Example 1. A549 cell culture

[0252] DMEM / F-12 basic medium is added with 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin as the culture environment for the A549 cell line. The A549 cell line is cultured in a humidified incubator at an ambient temperature of 37°C and a carbon dioxide content of 5%. During the experiment, fresh cells are taken and washed with 1X phosphate buffered saline (DPBS) twice, and then the cells are suspended in 1X DPBS containing 0.04% bovine serum albumin.

[0253] Example 2. Preparation of mouse lung tissue single cell suspension

[0254] Prepare one 2-month-old, one 3-month-old, and one 5-month-old C57 / BJ6 male mouse each under specific pathogen-free (SPF) conditions with free feeding and drinking. The mice are placed in an IVC cage with a 12-hour light-dark cycle and fed with Co60-irradiated laboratory feed. The animals are euthanized with carbon dioxide, and the lung tissue is collected. A single cell suspension is prepared using a tissue digestion kit. The cell suspension is lysed with ACK lysis solution, and dead cells are removed using a dead cell removal kit, and then filtered through a 40-μl filter screen. After centrifugation, the cells are resuspended in 1X PBS for use.

[0255] Example 3. Tissue or single cell suspension fixation preservation

[0256] DSP fixation solution was prepared according to the instructions of the dithiobis(succinimidyl propionate) (DSP) reagent.

[0257] A piece of tissue about 3x3 mm (the smaller the better) was immersed in 500 μl of fixation solution and incubated at room temperature for 45 minutes. For single cell suspension, cells were washed at least twice in pre-chilled PBS. Cells were pelleted, resuspended in 500 μl of fixation solution, and incubated at room temperature for 30 minutes.

[0258] Add 10 μl of 1 M Tris-HCl (pH-7.5) at room temperature, vortex mix, and let stand for at least 15 minutes at room temperature.

[0259] For tissue, tissue pieces were pelleted by centrifugation at 500x g for 20 minutes and supernatant was removed; for cells, vortex mix and centrifuge at 500x g for 5 minutes at room temperature to pellet the cells.

[0260] Tissue or cells were placed in DPBS and stored at 4 °C, or placed in DMEM medium containing 10% FBS and stored at -80 °C.

[0261] Example 4. PSCSR-seq library construction and sequencing

[0262] Cell suspension was stained with Hoechst-33342 and propidium iodide and incubated with ReadyProbes Cell Viability Imaging Kit (R37610, Thermo Fisher) at 37 °C in a thermostatic heater for 20 minutes, centrifuged at 300x g for 5 minutes, and resuspended the cells with 1 ml of lx DPBS containing 0.04% BSA. After cell counting, the cell suspension was diluted to a concentration of 1 cell / 35 nl with a mixture containing lx Second Diluent (640196, Takara) and 0.4 u (u represents enzyme activity unit) Recombinant Ribonuclease Inhibitor (N2515, Promega). The cell suspension was divided into 5184 microwells of a SMARTer icell8 350v chip (640019, Takara). All microwells of the ICCELL8 chip were imaged with a fluorescence microscope (Olympus BX43) and analyzed using CellSelect software (Takara) to determine the number of live cells and cells. After single cell sorting, the chip was transferred to a -80 °C freezer for 1 hour of freezing.

[0263] Chips were incubated on PCR machine (Bio-Rad) for 5 min at 25 °C, 5 min at 75 °C and immediately placed on ice. A mix of 168 μΐ of 2 uM sRNA 3’ adapter (RA3-A2N), 16 U T4 RNA Ligase 2, truncated KQ (M0373S, NEB), 2X T4 RNA ligase buffer, 1 U Ribonuclease Inhibitor, 0.2% triton x-100 (T9284, Sigma-Aldrich) was prepared and dispensed into selected microwells using the MSND system. Chips were incubated for 6 h at 25 °C and 8-10 h at 4 °C.

[0264] A mix of 168 μΐ of 2 uM mRNA reverse transcription primer (MM-T50T) with more than 40 T’s and 5X RT buffer was prepared and dispensed into selected microwells using the MSND system. Chips were heated for 3 min at 95 °C and placed on ice. A mix of 168 μΐ of mRNA reverse transcription reaction (2 mM dNTP, 12 U Maxima H Minus Reverse Transcriptase [EP0753 Thermo Fisher], 4 uM strand displacement primer [TSO-5TL], 2 U Ribonuclease Inhibitor) was prepared and dispensed into selected microwells using the MSND system. Chips were placed on PCR machine and incubated for 90 min at 50 °C and 5 min at 85 °C.

[0265] The sRNA reverse transcription mix (15 uM tagged reverse transcription primer [SCSR-RTP], 2.5x Lambda Exonuclease buffer) was added to the A5-P8, A9-d9, A10-D10 wells of the 384 well plate in a 20 ul volume per well. The "index 1" program was executed, dispensing 35 nl of tagged reverse transcription primer to the selected microwells. The chip was placed on a PCR machine for 2 minutes at 70 °C, then left on ice. The 168 ul ligation removal reaction (2.4U Lambda Exonuclease [EN0562, Thermo Fisher], 5U 5' Deadenylase [M0331, NEB], 1.2U Ribonuclease Inhibitor) was prepared and added to the M3-P3 wells of the 384 well plate, dispensed to the selected microwells using the MSND system. The chip was centrifuged and placed in a PCR machine for 30 minutes at 30 °C, 60 minutes at 37 °C, then 10 minutes at 75 °C.

[0266] The 168 ul 2uM sRNA 5' adaptor (SR5T-UUG) and 7mM ATP were incubated at 70 °C for 2 minutes, then added to the sRNA 5' ligation reaction (5.6U T4 RNA Ligase 1 (ssRNA Ligase), High Concentration [M0437M, NEB], 3X T4 RNA ligase buffer, 1.4U Ribonuclease Inhibitor). The mix was added to the M11-P11 wells of the 384 well plate, dispensed to the selected microwells using the MSND system. Incubated at 25 °C for 2 hours, 15 minutes at 65 °C.

[0267] The 168 ul sRNA reverse transcription reaction (1X First-strand buffer, 48mM DTT, 1.6mM dNTP [4019, Takara], 1.6U Ribonuclease Inhibitor, 22.4U Superscript III reverse transcriptase [18080-085, Thermo Fisher]) was prepared and added to the A19-D19 wells of the 384 well plate, dispensed to the selected microwells using the MSND system. Incubated at 52 °C for 50 minutes, 15 minutes at 70 °C.

[0268] Mix 6.3 uM tagged PCR-1 primer (SR5T-P1) with 3.3X PCR buffer, 0.2U Phanta HS Super-Fidelity DNA Polymerase (P502-d1, Vazyme), add to 384-well plate (position A13-P16, A17-d17, A18-D18), 20ul volume per well. Inject 35nl PCR-1 mixture into microwells with "index-2" program. Place the chip on a PCR machine, react at 95°C for 3 minutes, then 12-14 cycles of 95°C for 30 seconds, 65°C for 30 seconds, 72°C for 1 minute, finally at 72°C for 5 minutes.

[0269] After reaction, the chip is inverted and centrifuged at 3000xg for 10 minutes, and the product is collected into a centrifuge tube. The collected PCR-1 product is purified and recovered with 1.8x nucleic acid purification magnetic beads (Ampure XP, A63882, Beckman Coulter or SPRI, B23319, Beckman Coulter or other company's alternative products). The product fragment size distribution is detected with an Agilent 2100 Bioanalyzer, and quantified with a Qubit dsDNA HS assay kit (Q32854, Thermo Fisher). The product is recovered by fragment screening with Pippin Prep, and the sRNA library recovery range is 134-162bp, and the mRNA library recovery range is 270-650bp.

[0270] The recovered DNA is used as a template for PCR-2 reaction. The 40ul PCR reaction system is: 0.2mM dNTP, 1x PCR buffer, 0.02U Phanta Max Super-Fidelity DNA Polymerase (P505-d1, Vazyme), 0.2uM SCSR-PCR-1 primer, 0.2uM SCSR-PCR-2 primer, and the reaction conditions are: 95°C for 3 minutes, then 6-12 cycles of 95°C for 30 seconds, 65°C for 30 seconds, 72°C for 1 minute, finally at 72°C for 5 minutes. The PCR-2 product is purified and recovered with 1.6x nucleic acid purification magnetic beads.

[0271] The content of the PSCSR-seq library is measured with a Qubit high sensitivity kit, the size distribution is measured with an Agilent 2100 Bioanalyzer, and the quality is detected with a library quantification kit (KK4824, Kapa). The sample is sequenced with an Illumina HiSeq2500 and HiSeq X Ten instrument.

[0272] Comparative Example 1

[0273] In the mRNA reverse transcription reaction, in step 4, Maxima HMinus reverse transcriptase and universal reverse transcriptase SMARTScribe TM Reverse transcriptase, under other conditions, compare the effects of two reverse transcriptases on RNA detection. Other reaction steps remain unchanged. Compare the effects of using different reverse transcriptases on the detected mRNA species and miRNA species. The results are shown in Figure 4 .

[0274] It is found that the reverse transcriptase used in the present application has better results in detecting RNA species than the existing universal strand displacement reverse transcriptase.

[0275] Comparative Example 2

[0276] Comparative experiments were conducted between the co-sequencing technology of the present application and the co-sequencing technology of Non-Patent Document 1, and the results are shown in Figure 5 .

[0277] It can be seen that the single cell mRNA and sRNA co-sequencing technology of the present application can detect more than twice the number of miRNA species of other similar methods under the same sequencing depth of a single cell, and at the same time has the sensitivity of mRNA matching other methods.

[0278] Comparative Example 3

[0279] In the mRNA reverse transcription reaction, due to the characteristics of the reverse transcriptase, the reverse transcription primer and the strand displacement primer produce non-specific binding to obtain certain by-products. If the universal reverse transcription primer (usually the number of T is 18-25) is designed, the length of the by-product will coincide with the length of the sRNA product, resulting in the inability to remove the by-product by fragment screening. Therefore, we designed a longer reverse transcription primer to ensure that the by-product of the mRNA library can be effectively removed. As shown in Figure 6 , we compared different reverse transcription primers and found that when the number of T exceeds 40, the proportion of by-products decreases significantly, and there is no significant difference in gene alignment rate. In addition, we compared the universal design scheme at the 3' end of the reverse transcription primer (3' end is VN) and the present application (3' end is T, as shown in SEQ ID NO: 3), and found that the gene alignment rate obtained by the present application is significantly higher than that of the universal design scheme. Further in single cell experiment Figure 7 ), it is found that the present application (the number of T in the reverse transcription primer is more than 40, as shown in SEQ ID NO: 3) has no significant difference in gene alignment rate and gene number detection with 40T, but the by-product produced by the present application is significantly less than that of 40T, proving that our optimized primer design is effective in single cell analysis.

[0280] The main reagents involved in the present application and their suppliers and specifications are shown in Table 1.

[0281] Table 1

[0282]

[0283]

Claims

1. A method for co-sequencing single-cell mRNA and sRNA, comprising the following steps: (1) Prepare a single-cell suspension, add it to the micropores of the chip of the single-cell operating system, and select a single live cell micropore for the experiment; (2) Cell lysis reaction; (3) sRNA 3' end ligation reaction; (4) mRNA reverse transcription reaction; (5) Remove sRNA free adapter reaction; (6) sRNA 5' end ligation reaction; (7) sRNA reverse transcription reaction; (8) For the first PCR reaction, purify and recover the PCR-1 product, divide the PCR-1 product into two equal parts, and screen and recover the mRNA and sRNA libraries respectively. (9) The second PCR reaction is carried out by using the screened and recovered PCR-1 product as a template, and then purifying and recovering the PCR-2 product to obtain a single-cell mRNA and sRNA co-sequencing library that can be directly used for sequencing.

2. The method according to claim 1, wherein, In the sRNA 3' end ligation reaction in step (3), the nucleotide sequence of the 3' adapter used is shown in SEQ ID NO: 1; In the sRNA 5' ligation reaction in step (6), the nucleotide sequence of the 5' adapter used is shown in SEQ ID NO:

2.

3. The method according to claim 1, wherein, Step (2) involves transferring the chip to a -80°C freezer for 1 hour and then incubating it at 25°C for 5 minutes until it reaches room temperature.

4. The method according to claim 1, wherein, Step (3) involves adding the sRNA 3' end ligation reaction mixture into the selected microwells, then transferring the chip to the PCR instrument and executing the sRNA 3' end ligation reaction program; Preferably, the sRNA 3' end ligation reaction mixture comprises a 3' adapter, T4 RNA ligase 2, T4 RNA ligation buffer, and a ribonuclease inhibitor; preferably, the sRNA 3' end ligation reaction mixture further comprises Triton X-100 lysis buffer and a ribonuclease inhibitor; Preferably, the sRNA 3' end ligation reaction procedure is incubation at 25°C for 6 hours, followed by reaction at 4°C for 8-10 hours; Preferably, before performing the sRNA 3' end ligation reaction procedure, the chip is further heated at 75°C for 5 minutes.

5. The method according to claim 1, wherein, Step (4) involves adding the mRNA reverse transcription mixture into the selected microwells, then adding the reverse transcription reaction solution, and then transferring the chip to a PCR instrument to execute the reverse transcription reaction program. Preferably, the mRNA reverse transcription mixture includes mRNA reverse transcription primers and reverse transcription buffer; Preferably, the mRNA reverse transcription reaction solution comprises dNTPs, reverse transcriptase, strand displacement primers, and ribonuclease inhibitors; Preferably, the reverse transcriptase includes Maxima H Minus reverse transcriptase and SuperScript. TM II Reverse Transcriptase, SuperScript TM IV reverse transcriptase, Maxima reverse transcriptase, SMARTScribe TM Reverse transcriptase; Preferably, the mRNA reverse transcription primer is shown in SEQ ID NO: 3; The sequence of the strand substitution primer is shown in SEQ ID NO: 4; Preferably, the reverse transcription reaction procedure is to react at 50°C for 90 minutes, followed by heating at 85°C for 5 minutes.

6. The method according to claim 1, wherein, Step (5) involves removing the free adapter from sRNA by adding the adapter removal reaction solution into the selected microwells, then transferring the chip to a PCR instrument and executing the adapter removal reaction program. Preferably, the components of the adapter removal reaction solution include exonuclease, 5' deadenylase, and ribonuclease inhibitor; Preferably, the connector removal reaction procedure is to incubate at 30°C for 30 minutes, then at 37°C for 60 minutes, and finally at 75°C for 10 minutes. Preferably, before performing the free adapter removal reaction, the method further includes adding the sRNA reverse transcription mixture into the selected microwells, and then transferring the chip to a PCR instrument for a heated reaction; Preferably, the components of the sRNA reverse transcription mixture include tagged sRNA reverse transcription primers and exonuclease buffer; The tagged sRNA reverse transcription primers include one or more as shown in SEQ ID NO: 5 to SEQ ID NO: 76; The heating reaction was carried out at a temperature of 70°C for 2 minutes.

7. The method according to claim 1, wherein, Step (6) involves adding the 5' ligation reaction solution to the selected microwells, then transferring the chip to the PCR instrument and executing the 5' ligation reaction program for the sRNA. Preferably, the sRNA 5' end ligation reaction solution comprises a 5' adapter, T4 RNA ligase 1, T4 RNA ligation buffer, ATP, and a ribonuclease inhibitor; Preferably, the procedure for the sRNA 5' ligation reaction is to incubate at 25°C for 2 hours, followed by heating at 65°C for 15 minutes.

8. The method according to claim 1, wherein, Step (7) involves adding the sRNA reverse transcription reaction solution into the selected microwells, then transferring the chip to a PCR instrument to execute the sRNA reverse transcription reaction program. Preferably, the components of the sRNA reverse transcription reaction solution include a single-strand synthesis buffer, DTT, dNTPs, a ribonuclease inhibitor, and reverse transcriptase; Preferably, the sRNA reverse transcription reaction procedure is to react at 52°C for 50 minutes, followed by heating at 70°C for 15 minutes.

9. The method according to claim 1, wherein, The first PCR reaction in step (8) involves adding the PCR-1 reaction solution into the selected microwells, then transferring the chip to the PCR instrument and executing the PCR-1 reaction program. Preferably, the PCR-1 reaction solution comprises tagged PCR-1 primers, dNTPs, PCR buffer, and DNA polymerase; The sequences of the tagged PCR-1 primers include one or more as shown in SEQ ID NO: 77 to SEQ ID NO: 148; Preferably, the PCR-1 reaction program is as follows: 95°C for 3 minutes, then 12-14 cycles of 95°C for 30 seconds, then 65°C for 30 seconds, then 72°C for 1 minute, and finally 72°C for 5 minutes.

10. The method according to claim 1, wherein, Step (8) Purify the PCR-1 product using nucleic acid purification magnetic beads, and then perform fragment screening and recovery on the PCR-1 product; Preferably, the product fragment size distribution is detected using an Agilent 2100 bioanalyzer, followed by quantification using a Qubit dsDNA HS detection kit, and product fragments are screened using Pippin Prep. Preferably, the recovery range of the sRNA library is 134-162 bp, and the recovery range of the mRNA library is 270-650 bp; More preferably, the second PCR reaction is performed by using the recovered PCR-1 product as a template to prepare the PCR-2 reaction solution and execute the PCR-2 reaction procedure. Preferably, the PCR-2 reaction solution comprises SCSR-PCR-1 primers, tagged SCSR-PCR-2 primers, dNTPs, PCR buffer, and DNA polymerase. The sequence of the SCSR-PCR-1 primer is shown in SEQ ID NO: 149, and the sequence of the tagged SCSR-PCR-2 primer includes one or more of the primers shown in SEQ ID NO: 150 to SEQ ID NO: 161; Preferably, the PCR-2 reaction program is as follows: react at 95°C for 3 minutes, then for 6-12 cycles of reacting at 95°C for 30 seconds, reacting at 65°C for 30 seconds, reacting at 72°C for 1 minute, and finally reacting at 72°C for 5 minutes. Preferably, the mRNA PCR-2 product is purified using nucleic acid purification magnetic beads (1.6x); Preferably, the content of the library is determined using the Qubit high-sensitivity kit, its size distribution is determined using an Agilent 2100 bioanalyzer, and its quality is detected using a library quantification kit. More preferably, the single-cell operating system is ICELL8; Preferably, the cells include A549 cells and single cells from mouse lung tissue.

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