Method for constructing single cell sequencing library and use thereof

Abstract

Provided in the present invention is a method for constructing single cell sequencing libraries, comprising the following steps: a) lysing a single cell to obtain a single cell lysate; b) separating the nucleus and the cytoplasm in the single cell lysate obtained in step a) to obtain a nuclear solution and a total RNA solution; and c) constructing a chromatin DNA library with the nuclear solution obtained in step b) to obtain a chromatin-accessibility sequencing library of the single cell; and constructing a transcriptome library with the total RNA solution obtained in step b) to obtain a transcriptome sequencing library of the single cell.

Claims

1. A method for constructing single-cell sequencing libraries, comprising the steps of: a) conducting a first lysis of a single cell to obtain a first single-cell lysate; b) segmenting the nucleus and the cytoplasm in the first single-cell lysate obtained in step a) to obtain a nucleus-containing solution and a total RNA-containing solution; c) constructing a chromatin DNA library with the nucleus-containing solution obtained in step b) to obtain a chromatin accessibility sequencing library of the single cell; and constructing a transcriptome library with the total RNA-containing solution obtained in step b) to obtain a transcriptome sequencing library of the single cell, wherein, in step c), the chromatin DNA library is constructed by digesting with Tn5 transposase, wherein the construction of the chromatin DNA library by using Tn5 transposase includes the steps of: c1) cutting the open chromatin regions with Tn5 transposase, comprising fragmenting the chromatin DNA with Tn5 transposase, followed by terminating the fragmentation; and c2) performing a second lysis of the product obtained in step c1); and then conducting a first amplification of the fragmented chromatin DNA.

2. The method according to claim 1, wherein the second lysis is carried out by using RLT Plus buffer.

3. The method according to claim 2, wherein the construction of the chromatin DNA library by using Tn5 transposase further includes: a second amplification of the first amplification product obtained in step c2).

4. The method according to claim 3, wherein a primer pair consisting of the nucleotide sequence 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO: 1)-3′ and the nucleotide sequence 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(SEQ ID NO: 2)-3′ is used in the first amplification in step c2); and a primer pair consisting of the nucleotide sequence 5′-phos-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC(SEQ ID NO: 3)-3′ and the nucleotide sequence 5′-TGTGAGCCAAGGAGTTGTTGTCTTC(SEQ ID NO: 4)-barcode sequence-GTCTCGTGGGCTCGG(SEQ ID NO: 5)-3′ is used in the second amplification.

5. The method according to claim 3, wherein a step of determining the number of amplification cycles required for the second amplification by using a real-time fluorescent quantitative PCR is further comprised between the first amplification of step c2) and the second amplification.

6. The method according to claim 5, wherein the step of determining the number of amplification cycles required for the second amplification comprises: performing a real-time fluorescent quantitative PCR by using the first amplification product obtained in step c2) as a template and using a primer pair consisting of the nucleotide sequence 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO: 1)-3′ and the nucleotide sequence 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(SEQ ID NO: 2)-3′, and finding the number of cycles corresponding to ⅓ of the plateau fluorescence intensity in the resulting linear amplification curve, which is the number of amplification cycles required for the second amplification.

7. The method according to claim 3, wherein the constructed chromatin DNA library is amplified after completion of the construction of the chromatin DNA library.

8. The method according to claim 2, wherein a carrier DNA is added to the system in the process of the second lysis.

9. The method according to claim 8, wherein the carrier DNA is added in an amount of 4-6 ng per μl of the the system volume.

10. The method according to claim 1, wherein a single fragmentation system used for fragmenting the chromatin DNA in step c1) comprises: the nucleus-containing solution; 5× fragmentation buffer, 0.2 μl a per μl of the reaction system volume; TTE Mix V5S, 0.03-0.05 μl per μl of the reaction system volume; Tris-HCl, pH 7.5, 8-12 mM; NaCl, 8-12 mM; with the balance being water.

11. The method according to claim 1, wherein the construction of the transcriptome library includes: c1′) reverse transcribing the total RNA into cDNA, and amplifying the cDNA to obtain a cDNA amplification product; c2′) fragmenting the cDNA amplification product obtained in step c1′) with Tn5 transposase, and amplifying the resulting fragmented product to obtain a transcriptome sequencing library of the single cell.

12. The method according to claim 1, wherein both the constructions of the chromatin DNA library and the transcriptome library in step c) are carried out in a microliter-scale reaction system.

13. The method according to claim 1, wherein a single first lysis system used for the first lysis of the single cell in step a) comprises: TABLE-US-00024 Single-cell suspension ≥0.5 μl; NP-40 0.1-0.3% Tris-HCl, pH 7.5 8-12 mM; NaCl 8-12 mM; RNase inhibitor l-2 U/μl; with the balance being water.

14. A method for analysis of single-cell multi-omics, comprising: subjecting the chromatin accessibility sequencing library and the transcriptome sequencing library constructed by the method according to claim 1 to a high-throughput sequencing to obtain information on the chromatin accessibility and on the transcriptome sequence of the single cell, respectively; and performing a bioinformatics analysis on the obtained information on the chromatin accessibility and on the transcriptome sequence of the single cell.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an example of determining the number of amplification cycles required for the second amplification for a chromatin DNA sample by fluorogenic quantitative PCR.

(2) FIG. 2 shows an example of the amplification result of the fragmentation product of a chromatin DNA sample.

(3) FIG. 3 shows an example of the amplification result of a cDNA sample.

(4) FIG. 4 shows an example of the amplification result of the fragmentation product of a cDNA sample.

DETAILED DESCRIPTION

(5) To facilitate an understanding of the present invention, the following examples are enumerated. It should be understood by those skilled in the art that these examples are only intended to facilitate the understanding of the present invention but should not be construed as any limitation of the present invention.

(6) Example Construction of Single-Cell Libraries of 119 ES Cell Line

(7) In this example, a simultaneous construction method of single-cell chromatin accessibility libraries and transcriptome sequencing libraries according to the present invention was carried out by taking H9 ES cell line as an example to prepare single-cell libraries. Specific steps were as follows:

(8) 1. Preparation and Lysis of a Single Cell

(9) 1.1. Preparation of a Single Cell

(10) The adhered H9 ES cells were digested with Accutase. 1 ml of suspension of the digested cells was taken and centrifuged at 1000 rpm for 5 min, and the cell pellet was resuspended in 1×PBS, repeating the above once. The resuspended cells were diluted to a suitable density, and a single cell (volume less than 0.5 μl) was aspirated by using a glass needle having a diameter less than 200 μm to prepare a cell sample. 100 cells were used as a positive control and PBS was used as a negative control.

(11) 1.2. Lysis of a Single Cell

(12) A single-cell lysis system was prepared according to Table 1. 6.5 μl of lysis buffer was added to each single-cell suspension sample (0.5 μl), and mixed by flicking the tube wall gently. The mixture was centrifuged for 3 s, and transferred to a thermal cycler to react at 4° C. for 30 min for cell lysis.

(13) TABLE-US-00005 TABLE 1 Reagent Volume (μl) Single-cell suspension 0.5 10% NP-40 (Sigma, Cat. No. I8896) 0.07 1M Tris-HCl (pH 7.5) (Invitrogen) 0.07 1M NaCl (Sigma) 0.07 RNAse inhibitor (40 U/μl) (Takara, 2313A) 0.25 Nuclease-free water (Ambion AM9932) 6.04 Total 7

(14) 2. Cell Nucleus/RNA Separation

(15) 2.1 After the lysis, the tube was vortexed for 1 min, and centrifuged at 4° C. at 1000 g for 5 min;

(16) 2.2 4 μl of supernatant was carefully pipetted into a new PCR tube. 3 μl of the remaining liquid was a nucleus-containing solution. The RNA was in the supernatant.

(17) 3. Nucleus and chromatin DNA library construction with Tn5

(18) 3.1 Cutting the open chromatin regions with Tn5 3.1.1 5× Tagament Buffer L was thawed at room temperature and mixed by turning upside down for use. 3.1.2. A Tn5-based fragmentation reaction system was prepared according to Table 2, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(19) TABLE-US-00006 TABLE 2 Volume Reagent (μl) DNA solution (the nucleus-containing solution obtained in step 3 2.2) 5x Tagament buffer L (5x fragmentation buffer, BGE005B01) 1.4 TTEMix V5S 0.3 (Tn5 enzyme V5S, BGE005S, for fragmentation) 1M Tris, pH 7.5 (Invitrogen) 0.07 1M NaCl (Sigma) 0.07 Nuclease-free water (Ambion) 2.16 Total 7 3.1.3 The tube was transferred to a thermal cycler to react at 37° C. for 30 min. 3.1.4 Reagents for terminating the Tn5-based fragmentation were prepared according to Table 3, and mixed uniformly. 3.5 μl of the reagents for terminating the fragmentation were added to the fragmented product obtained as above, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(20) TABLE-US-00007 TABLE 3 Volume Reagent (μl) 0.1M EDTA, pH 8.0 (Ambion) 2.1 0.1M Tris, pH 8.0 (Ambion) 0.42 Nuclease-free water (Ambion) 0.98 Total 3.5 3.1.5 The tube was transferred to a thermal cycler to react at 50° C. for 30 min.

(21) 3.2 Second lysis and addition of a carrier DNA 3.2.1 Reagents for the second lysis were prepared according to Table 4 and mixed uniformly. To the product obtained in step 3.1.5, 6 μl of the reagents were added (with a total volume of 16.5 μl), centrifuged for 3 s, and left at room temperature for 15 min.

(22) TABLE-US-00008 TABLE 4 Volume Reagent (μl) RLT Plus buffer (Qiagen, Cat. No. 1053393) 3 10 ng/μl of the carrier DNA 3 Total 6 In Table 4, the carrier DNA was a foreign DNA with a fragment size of between 1 kb and 10 kb and being heterologous to the genome of the cell of interest. It may be linear or circular. For example, a DNA fragment derived from bacteria satisfying the requirements, or a conventional plasmid cloning carrier can be used as a carrier DNA. 3.2.2 Sterilized ultrapure water was added to 40 μl, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(23) 3.3 DNA purification

(24) In order to avoid interference from other substances in the subsequent PCR reaction system, DNA purification was carried out in this step. Specifically, purification was carried out by using Agencourt AMPure XP magnetic beads (1.8×) and finally the DNA was dissolved in 9 μl of sterilized ultrapure water.

(25) 3.4 First DNA amplification 3.4.1 Reagents for the first amplification were prepared according to Table 5 and mixed uniformly. 11 μl of the reagents for the first amplification were added to the purified product obtained in step 3.3, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(26) TABLE-US-00009 TABLE 5 Volume Reagent (μl) High-Fidelity 2X PCR Master Mix 10 (NEB, Cat. No. M0541L) Primer B (20 μM) 0.5 Primer C (20 μM) 0.5 Total 11 Wherein primer B had the sequence (as shown in SEQ ID NO: 1) of:

(27) TABLE-US-00010 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′;  Primer C had the sequence (as shown in SEQ ID NO: 2) of:

(28) TABLE-US-00011 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ 3.4.2 The tube was transferred to a thermal cycler to undergo the following procedure, 72° C. 5 min; 98° C. 1 min; 98° C. 15 s, 63° C. 30 s, 72° C. 1 min, 8 cycles. 3.4.3 Purification was carried out by using Agencourt AMPure XP magnetic beads (1.8×), and finally the DNA was dissolved in 20 μl of sterilized ultrapure water.

(29) 3.5 Determination of the cycle numbers of PCR required for the second amplification by using real-time fluorescent quantitative PCR 3.5.1 4 μl of the purified product obtained in step 3.4.3 was transferred into a new PCR tube. 3.5.2 Reagents for the amplification were prepared according to Table 6 and mixed uniformly. 16 μl of the reagents for the amplification were added to the PCR tube containing the purified product obtained in step 3.5.1, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(30) TABLE-US-00012 TABLE 6 Volume Reagent (μl) 2x SYBR ® Premix Ex Taq ™ II 10 (Takara, Cat. No. RR820W) Primer B (20 μM) 0.5 Primer C (20 μM) 0.5 Fluorescent dye ROX 0.08 (Takara, Cat. No. RR820W) Nuclease-free water (Ambion) 4.92 Total 16 3.5.3 The tube was transferred to a fluorescence quantitative PCR instrument to undergo the following procedure, 72° C. 5 min; 98° C. 30 s; 98° C. 10 s, 63° C. 30 s, 72° C. 1 min, 40 cycles. 3.5.4 The number of cycles corresponding to ⅓ of the plateau fluorescence intensity in the linear amplification Rn/Cycle curve was the number N of cycles required for the second amplification, as shown in FIG. 1.

(31) 3.6 The second DNA amplification 3.6.1 Reagents for the second amplification were prepared according to Table 7 and mixed uniformly. 17.1 μl of the reagents for the second amplification were added to the purified product obtained in step 3.4.3, and 0.7 μl of barcode primer N7 was further added, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(32) TABLE-US-00013 TABLE 7 Volume Reagent (μl) High-Fidelity 2X PCR Master Mix 16.4 (NEB, Cat. No. M0541L) N5 (25 μM) 0.7 Total 17.1 Wherein N5 primer had the sequence of:

(33) TABLE-US-00014 5′-phos-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC-3′ (i.e. SEQ ID NO: 3); N7 primer had the sequence of:

(34) TABLE-US-00015 5′-TGTGAGCCAAGGAGTTGTTGTCTTC(i.e. SEQ ID NO: 4)- barcode sequence-GTCTCGTGGGCTCGG(i.e. SEQ ID NO: 5)-3′.  Specifically, the barcode sequence may be 5′- ATTTATGACA-3′ (i.e. SEQ ID NO: 6). 3.6.2 The tube was transferred to a thermal cycler to undergo the following procedure, 72° C. 5 min; 98° C. 1 min; 98° C. 15 s, 63° C. 30 s, 72° C. 1 min, N cycles (N was the number of cycles as determined in 3.5.4). 3.6.3 Purification was carried out by using Agencourt AMPure XP magnetic beads (1×), and finally the DNA was dissolved in 20 μl of sterilized ultrapure water.

(35) 3.7 Product concentrations were determined by Qubit, and library construction results were tested with Agilent 2100. The desired library should be distributed above 100 bp, as shown in FIG. 2.

(36) Optionally, the constructed chromatin DNA library can be amplified with the specific amplification system as shown in Table 8:

(37) TABLE-US-00016 TABLE 8 Volume Reagent (μl) DNA solution (the solution obtained in 3.6.3) X (20 ng) 2x KAPA HiFi HotStart ReadyMix 25 (KAPA BIOSYSTEMS, Cat. No. KK2602) Primer 1 (20 μM) 1.5 Primer 2 (20 μM) 1.5 Nuclease-free water (Ambion) 22-X Total 50 Wherein primer 1 had the sequence of:

(38) TABLE-US-00017 5′-phos-GAACGACATGGCTACGATCCGACTT-3′ (the  underlined sequence was SEQ ID NO: 7); Primer 2 had the sequence of:

(39) TABLE-US-00018 5′-TGTGAGCCAAGGAGTTGTTGTCTTC-3′ (i.e. SEQ ID NO: 4).

(40) 3.8 Further, pre-processing and sequencing were performed based on a specific sequencing platform.

(41) 4. Preparation of a cDNA sample from the total RNA, fragmentation of the cDNA sample, and library construction.

(42) 4.1 Reverse transcription of the total RNA to cDNA

(43) Names and Sequences of the Reference Primers:

(44) Oligo-dT:

(45) 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′ (the underlined sequence was SEQ ID NO: 8);

(46) Template-Switching Oligo (LNA):

(47) 5′-AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3′ (wherein the underlined sequence was SEQ ID NO: 9, the rG was an RNA base, and the +G was a locked nucleic acid (LNA) modification). 4.1.1 1 μl of oligo-dT (10 μM) and 1 μl of dNTP (10 mM) (ENZYMATICS) were added to the pipetted RNA supernatant in step 2.2, mixed by flicking the tube wall gently, and centrifuged for 3 s. The tube was transferred to a thermal cycler to react at 72° C. for 3 min. After the reaction, materials were centrifuged to the bottom of the tube, and placed on ice for use. 4.1.2 A reverse transcription system was prepared according to Table 9, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(48) TABLE-US-00019 TABLE 9 Volume Reagent (μl) RNA solution (the solution obtained in step 6 4.1.1) SuperScript II reverse transcriptase 200 U/μl 0.75 (Invitrogen, Cat. No. 18064) RNAse inhibitor 40 U/μl (Takara) 0.375 5xSuperscript II First-Strand Buffer 3 (Invitrogen, Cat. No. 18064) 100 mM DTT (Invitrogen) 0.75 5M Betaine (SIGMA, Cat. No. B0300-1VL) 3 0.2M MgCl.sub.2 (MILLIPORE) 0.45 100 μM Template-Switching Oligo (LNA) 0.15 (EXIQON,Cat. No. 500100) Nuclease-free water (Ambion) 0.525 Total 15 4.1.3 The tube was transferred to a thermal cycler to undergo the following procedure, 42° C. 90 min; 50° C. 2 min, 42° C. 2 min, 10 cycles; 72° C. 5 min.

(49) 4.2 cDNA amplification

(50) Name and Sequence of the Reference Primer

(51) TABLE-US-00020 IS primer:  (SEQ ID NO: 10) 5′-AAGCAGTGGTATCAACGCAGAGT-3′.  4.2.1 A cDNA amplification system was prepared according to Table 10, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(52) TABLE-US-00021 TABLE 10 Volume Reagent (μl) cDNA solution (the product obtained in step 15 4.1.3) 2x KAPA HiFi HotStart Ready Mix 14.7 (KAPA BIOSYSTEMS, Cat. No. KK2602) 10 μM IS Primer 0.3 Total 30 4.2.2 The tube was transferred to a thermal cycler to undergo the following procedure, 98° C. 3 min; 98° C. 20 s, 67° C. 20 s, 72° C. 6 min, 20 cycles; 72° C. 5 min. 4.2.3 cDNA Purification Purification was carried out by using Agencourt AMPure XP magnetic beads. 4.2.4 The cDNA amplification results were detected by Agilent 2100, as shown in FIG. 3.

(53) 4.3 cDNA library construction with Tn5 4.3.1 cDNA fragmentation 4.3.1.1 5× Tagament Buffer L was thawed at room temperature and mixed by turning upside down for use. 4.3.1.2 A Tn5 fragmentation system was prepared according to Table 11. The components were mixed thoroughly by gently pipetting 20 times with a pipette, and centrifuged for 3 s.

(54) TABLE-US-00022 TABLE 11 Volume Reagent (μl) 1 ng/μl cDNA (the purified product obtained in step 2 4.2.3) 5x Tagment Buffer L 2 (5x fragmentation buffer, BGE005B01) Tagment Enzyme Advanced Mix V5S 0.8 (Tn5 enzyme V5S, BGE005S, for fragmentation) Nuclease-free water (Ambion) 5.2 Total 10 4.3.1.3 The PCR tube was incubated in a thermal cycler at 55° C. for 10 min, and was taken out when the sample temperature dropped to 4° C. 4.3.1.4 2.5 μl of 5×NT Solution was added to the product obtained in step 4.3.1.3, mixed thoroughly by gently pipetting 20 times with a pipette, and left at room temperature for 5 min. 4.3.2 Amplification of the fragmented cDNA product Names and sequences of the reference primers: Pimer 1, primer 2, N5 primer, and N7 primer, with the sequences as described above, were used for the amplification in this step. 4.3.2.1 A reaction system for amplifying the product of the cDNA fragmentation was prepared according to Table 12, mixed by flicking the tube wall gently, and centrifuged for 3 s.

(55) TABLE-US-00023 TABLE 12 Volume Reagent (μl) cDNA solution (the product obtained in step 12.5 4.3.1.4) Primer 1 (10 μM) 1 Primer 2 (10 μM) 1 Primer N5 (0.5 μM) 1 Primer N7 (0.5 μM) 1 2x KAPA HiFi HotStart Ready Mix 25 Nuclease-free water (Ambion) 8.5 Total 50 4.3.2.2 The tube was transferred to a thermal cycler to undergo the following procedure, 72° C. 5 min; 95° C. 3 min; 98° C. 20 s, 60° C. 15 s, 72° C. 30 s, 16 cycles; 72° C. 5 min. 4.3.2.3 The PCR product was selectively purified by using Agencourt AMPure XP magnetic beads. 4.3.2.4 Product concentrations were determined by Qubit, and library construction results were tested with Agilent 2100. The desired library had a main peak of between 150 and 350 bp, as shown in FIG. 4.

(56) 5. High-throughput sequencing and data analysis

(57) The libraries constructed as above can be sequenced by using a next-generation sequencing platform which is currently mainstream (e.g., BGISEQ-500, Hiseq2000, Hiseq4000, etc.). The sequencing platform used in this example was BGISEQ-500. The post-sequencing analysis included separate filtering of single-cell accessibility and transcriptome data, data alignment, and downstream mining and analysis of personalized data.

(58) The applicant declares that methods of the present invention and use thereof have been demonstrated through the above embodiments, and however, the present invention is not limited thereto. It should be apparent to those skilled in the art that, for any improvement of the present invention, the equivalent replacement and addition of the parameters or steps involved in the methods of the present invention, and the selection of specific modes, etc., will all fall within the protection scope and the disclosure scope of the present invention.