WO2022237035A1 - 一种超高通量单细胞测序方法 - Google Patents

一种超高通量单细胞测序方法 Download PDF

Info

Publication number
WO2022237035A1
WO2022237035A1 PCT/CN2021/119166 CN2021119166W WO2022237035A1 WO 2022237035 A1 WO2022237035 A1 WO 2022237035A1 CN 2021119166 W CN2021119166 W CN 2021119166W WO 2022237035 A1 WO2022237035 A1 WO 2022237035A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
cell
bridging
molecular marker
cells
Prior art date
Application number
PCT/CN2021/119166
Other languages
English (en)
French (fr)
Inventor
郭国骥
廖原
陈海德
韩晓平
王晶晶
张国栋
杨蕾
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202110517750.6A external-priority patent/CN113106150B/zh
Priority claimed from CN202110911428.1A external-priority patent/CN113604545B/zh
Application filed by 浙江大学 filed Critical 浙江大学
Priority to US17/785,045 priority Critical patent/US20240182962A1/en
Publication of WO2022237035A1 publication Critical patent/WO2022237035A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • the invention relates to the technical field of single-cell sequencing, in particular to an ultra-high-throughput single-cell sequencing method.
  • chromatin In the genome, most of the chromatin is tightly wound in the nucleus and is not transcriptionally active.
  • the chromatin state is dynamically regulated in a cell-type-specific manner, and partially dense chromatin becomes loose in a specific cell state. These loose chromatin are called open chromatin or accessible chromatin. ).
  • open chromatin or accessible chromatin By detecting the opening of cell chromatin, information about cell transcription regulation can be obtained, such as where transcription factors can bind to gene promoters, which genes in cells may be efficiently transcribed, etc.
  • Commonly used detection methods are ATAC-seq, DNase-seq, MNase-seq, FAIRE-seq and ChIP-seq, etc. These methods are based on different principles to interrupt and mark open chromatin regions.
  • Quantitative chromatin transposase accessibility sequencing method assay for transposase-accessible chromatin with high-throughput sequencing
  • uses modified Tn5 transposase which can randomly insert specific DNA sequences as transposons into open regions of chromatin, The sequence of the entire open region can be completely captured directly, so ATAC-seq is now widely used in the sequencing of open chromosomes.
  • the invention provides an ultra-high-throughput single-cell sequencing method, which can obtain the specific transcriptome information of millions of single cells at one time.
  • the present invention firstly provides an ultra-high-throughput single-cell sequencing method, comprising the following steps:
  • the molecular marker microbeads include a microbead body and a coupled molecular marker sequence, and the molecular marker sequence includes sequentially arranged:
  • a reverse transcription sequence for intracellular reverse transcription comprising sequentially arranged:
  • the second cell tag sequence is combined with the first cell tag sequence to form a cell tag sequence, and the cell tag sequence is used to identify the cell from which the mRNA corresponding to each sequence in the constructed sequencing library is obtained;
  • the molecular tag sequence is used to identify the mRNA corresponding to each sequence in the constructed sequencing library
  • Poly T tail for complementary pairing with mRNA with poly-A sequence in cells
  • a bridging primer used to connect the marker sequence in a) above to the reverse transcription sequence in b), the two ends of the bridging primer are respectively complementary to the first bridging sequence and the second bridging sequence paired sequence;
  • step (3) The cells after the intracellular reverse transcription in step (2) are placed in a separate space between a molecularly labeled microbead and one or more cells through microwell plate technology or microfluidic technology, and reacted in the lysate Cells were lysed, incubated, and the bridging primers were complementary paired with the first bridging sequence and the second bridging sequence respectively, and then ligated using ligase, so that the first bridging sequence and the second bridging sequence were connected to obtain microbead-coupled Molecular marker sequence-reverse transcription sequence-cDNA sequence;
  • step (4) Construct a cDNA sequencing library from the product obtained in step (4), and then perform high-throughput sequencing to obtain specific transcriptome information of millions of single cells.
  • the sequencing method is a transcriptome sequencing method.
  • the second cell label sequence is introduced in the reverse transcription sequence of each mRNA in the form of a reverse transcription sequence in the cell, thereby
  • a molecular marker bead binds multiple cells, it can be distinguished through this second cell label sequence, otherwise, only relying on the first cell label sequence on the molecular marker bead, it is impossible to distinguish the cells that bind to the same cell. Sequences of multiple cellular origins for molecularly labeled microbeads.
  • the microwells on the experimental microwell plate should be prepared as far as possible to accommodate only one molecular marker microbead and one cell ( In this case, the relative size of the molecular marker microbeads and the cells should not be too large, otherwise, it is not easy to achieve a molecular marker microbead binding to a cell), and it is necessary to control the cell dropout rate at a low level so that the cells are sufficiently dispersed
  • the final sequencing result will misjudge the sequence in this case as coming from the same cell.
  • the second cell label sequence cooperates with the first cell label sequence to form a cell label sequence, which increases the number of combinations of cell label sequences and can detect a larger number of cells at one time.
  • the present invention also provides an ultra-high-throughput single-cell sequencing method, comprising the following steps:
  • the molecular marker microbeads include a microbead body and a coupled molecular marker sequence, and the molecular marker sequence includes sequentially arranged:
  • the second cell tag sequence is combined with the first cell tag sequence to form a cell tag sequence, and the cell tag sequence is used to identify cells from which each sequence in the constructed sequencing library is derived;
  • Mosaic Ends sequence for binding with Tn5 transposase, said Mosaic Ends sequence is a double-stranded structure, wherein one strand is connected with the second cell tag sequence;
  • a bridging primer used to connect the molecular marker sequence in a) above to the specific molecular tag sequence in b), the two ends of the bridging primer have respectively the first bridging sequence and the second bridging sequence Sequences that are complementary to each other;
  • step (3) adding specific molecular tag transposase embedding complex to the nucleus extracted in step (2) to carry out transposition reaction;
  • Collect microbeads coupled with molecular marker sequence-specific molecular marker sequence-chromatin transposase accessibility genome sequence perform PCR amplification to obtain the first cell marker sequence, the second cell marker sequence and Chromatin transposase accessibility genomic sequences of specific molecular tag sequences;
  • step (5) The product obtained in step (5) is used to construct a chromatin accessibility sequencing library, and then high-throughput sequencing is performed to obtain genomic sequence information of chromatin transposase accessibility of millions of single cells.
  • the sequencing method is a chromatin transposase accessibility sequencing method.
  • the second cell label sequence is brought into the corresponding sequence of the genome during the Tn5 transposase transposition reaction, so that a molecular marker microbead appears
  • this second cell label sequence can be used to distinguish, otherwise, only relying on the first cell label sequence on the molecular label bead, it is impossible to distinguish multiple cells bound to the same molecular label bead. Sequence of cell origin.
  • the coupling method of the microbeads and the molecular marker sequence is as follows: the amino group is used to replace the hydroxyl group at the C6 position of the nucleotide at the 5' end of the molecular marker sequence, the surface of the microbead is modified with a carboxyl group, and the carboxyl group is coupled with the amino group through condensation. Since the molecular marker sequence is a single-stranded oligonucleotide, the hydroxyl group on the first nucleotide at the 5' end is replaced with an amino group, the surface of the microbead is modified with a carboxyl group, and the molecular marker sequence is coupled to the on the microbeads.
  • At least part of the molecular tag sequence is a randomly synthesized random sequence.
  • the first cell tag sequence includes a plurality of specific fragments
  • the second cell tag sequence includes at least one specific fragment
  • the specific fragments at different positions are selected from the same or different specific fragment libraries, so The first cell tag sequence and the second cell tag sequence use different arrangements and combinations of the specific fragments to identify cells.
  • the preparation method of the molecularly labeled microbeads comprises the following steps:
  • the primers used to synthesize molecular marker sequences are divided into multiple primers according to the number of specific fragments, each primer contains a specific fragment, and each primer has a complementary linker sequence for bridging connection, which corresponds to
  • the primer at the 5' end of the molecular marker sequence also includes the universal primer sequence, and the primer corresponding to the 3' end of the molecular marker sequence also includes the first bridging sequence;
  • the molecular marker sequence is: 5'-TTTAGGGATAACAGGGTAATAAGCAGTGG TATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNTCGGTGACACGATCGNNNNNNNTCGTCGGCAGCGTC-3', wherein N represents any one of A/T/C/G, which is randomly synthesized.
  • the reverse transcription sequence is: 5'-[phos]ACACTCTTTCCCTACACGACGNNNNNNNnnnnnnnnnTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3', wherein, the phosphorylation modification at the 5' end provides a phosphate group for the ligation reaction; N represents any of A/T/C/G One means random synthesis; n means any of A/T/C/G, means random synthesis; 3' V means any of A/C/G, V means random synthesis.
  • the specific molecular tag sequence is: 5'-ACACTCTTTCCCTACACGACGNNNNNNNNNNNAGATGTGTA TAAGAGACAG-3', wherein, N represents any one of A/T/C/G, which is randomly synthesized, and the Mosaic Ends sequence complementary to form a double strand is: 5 '-CTGTCTCTTATACACATCT-3'.
  • the bridging primer is: 5'-CGT CGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3', ddC is modified by dideoxycytidine. Or remove the ddC at the 3' end.
  • the 6 ⁇ N random sequence is used as the second cell label sequence
  • the 10 ⁇ N random sequence is used as the molecular label sequence.
  • one transposase complex in the specific molecular label transposase embedding complex carries two gene fragments, and the two gene fragments are the specific molecule Tag sequence, or one gene segment is the specific molecular tag sequence, and the other gene segment is a general-purpose sequence, and the general-purpose sequence includes:
  • the primer-binding sequence for amplification is used as the primer-binding region during PCR amplification
  • the Mosaic Ends sequence is used to combine with the Tn5 transposase, the Mosaic Ends sequence is a double-stranded structure, and one of the strands is connected to the primer-binding sequence for amplification.
  • the cell sample to be sequenced contains 2 or more types of cells.
  • the ultra-high-throughput single-cell sequencing method of this application can realize simultaneous sequencing of multiple cells.
  • the microbead body is a magnetic bead; the cell after intracellular reverse transcription (reverse transcription sequencing) or the nucleus after transposition reaction (chromatin transposase accessibility sequencing) is added to the microbead In the well plate, then add molecularly labeled microbeads.
  • the diameter of the microwell in the microwell plate is just enough to accommodate one molecularly labeled microbead and one or more cells/nuclei; the dropout rate of cells or cell nuclei added to the microplate is controlled
  • the hole drop rate of the molecularly labeled microbeads added to the microwell plate is greater than 99%.
  • the ultra-high-throughput single-cell sequencing method of this application can realize the combination of one molecularly labeled microbead with multiple cells, when the microbead body is a magnetic bead and the microwell plate method is used, the hole drop rate when the cells are added to the microwell plate can be achieved greatly improved.
  • the present invention can also be used in a single-cell sequencing platform of a microfluidic method, in addition to the single-cell sequencing in which the microbead body is a magnetic bead and a microwell plate method is used.
  • the depth of the microwells in the microwell plate is 30-160 ⁇ m, the diameter of the micropores is 20-150 ⁇ m; the diameter of the microbead body is 20-145 ⁇ m.
  • the preparation method of the microporous plate is: (1) etching micropores on the silicon chip as the initial mold; (2) pouring polydimethylsiloxane on the initial mold, and taking it off after molding Polydimethylsiloxane becomes the second mold with microcolumn; (3) pouring hot-melt mass volume ratio on the second mold is 4% ⁇ 6% agarose, after cooling and forming, take off the agar Sugar is the microplate.
  • the ultra-high-throughput single-cell sequencing method of the present invention can perform ultra-high-throughput sequencing of single cells, and the number of cells sequenced at one time can reach millions of single cells, greatly improving the throughput of single-cell sequencing.
  • Figure 1 is a schematic diagram of a honeycomb-arranged microporous plate.
  • Figure 2 is a flowchart for the preparation of molecularly labeled magnetic beads.
  • Figure 3 is a schematic diagram of cells falling into a microwell plate.
  • Fig. 4 is a flow chart for constructing a cDNA library, wherein the universal sequence includes the universal primer sequence described in Fig. 2, cell tag sequence 1, linker sequence 1, cell tag sequence 2, linker sequence 2, and cell tag sequence 3.
  • Figure 5 is a size distribution diagram of the prepared cDNA sequencing library fragments.
  • Fig. 6 is a comparison chart of human-mouse mixed cell grouping.
  • Figure 7 is a comparison chart of sequencing read length/gene in different lysates.
  • Figure 8 is a graph showing the results of tSNE analysis of mouse testicular cells.
  • Figure 9 shows the joint embedding of the adapter sequence and Tn5 transposase, in which the two adapter sequences respectively contain the specific molecular tag sequence and the universal sequence, and the adapter sequence is incubated with Tn5 naked enzyme to generate a Tn5 embedding complex. Buried complexes carry both specific molecular tag sequences and general-purpose sequences.
  • Figure 10 is a flowchart for constructing an ATAC library, wherein the universal sequence includes the universal primer sequence, cell tag sequence 1, linker sequence 1, cell tag sequence 2, linker sequence 2 and cell tag sequence 3 in Figure 1; the bridge sequence includes a bridge sequence 1 and bridging sequence 2.
  • Figure 11 is a size distribution diagram of the prepared gene sequencing library fragments.
  • Figure 12 is a comparison chart of human and mouse mixed cell groups.
  • Figure 13 is a graph showing the size distribution of DNA fragments in human and mouse samples.
  • Figure 14 is the peak annotation distribution map of human and mouse samples.
  • Figure 15 is a diagram of TSS enrichment in mice.
  • Figure 16 is the overlapping distribution of mouse single cell ATAC and population ATAC peak.
  • Figure 17 is a graph comparing the distribution of mouse single-cell ATAC and population ATAC reads on chromosome 8 at 0-25 Mbp.
  • Figure 18 is a flow chart of constructing an ATAC library by the Tn5 embedding method with both ends tagged. Label sequence 3.
  • the size of the microwell plate according to the scale of the experiment (for example, 500,000 human 293T cells and 500,000 mouse 3T3 cells each, the size of the well plate is 1.8cm ⁇ 1.8cm), and etch microwells on the silicon wafer as the initial mold. Cylinder type, in which the micropore depth is 60 ⁇ m, the micropore diameter is 50 ⁇ m, and the hole spacing is 70 ⁇ m.
  • PDMS polydimethylsiloxane
  • the magnetic beads were purchased from Suzhou Zhiyi Microsphere Technology Co., Ltd. (Product No. MagCOOH-20190725), the surface was coated with carboxyl groups, and the diameter was 45 ⁇ m.
  • the preparation process of molecularly labeled magnetic beads is shown in Figure 2, with a total of 4 steps:
  • 6 ⁇ N is the core sequence of the cell label sequence, and the core sequence corresponding to each magnetic bead is different, and the 6 ⁇ N sequences in the three sequences corresponding to the same magnetic bead are also different, because each site has A There are 4 choices of /T/C/G, so the sequence of 6 ⁇ N can have 4 6 choices.
  • N represents any one of A/T/C/G, which is randomly synthesized.
  • sequence of the molecular marker is as follows: 5'-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNNTCGTCGGCAGCGTC-3', which can be used for ultra-high-throughput single-cell sequencing.
  • ESC mouse embryonic stem cells
  • human embryonic kidney cells (293T) each 5 million slowly dropwise with 5-10ml methanol (-20°C pre-cooled) respectively, fix at -20°C for 30 minutes, and at the same time, aliquot the bridging primers 6.5 ⁇ l per well into eight tubes, then dispensed into a 96-well plate containing 0.5 ⁇ l reverse transcription primers, and left to mix to form a total of 1 ⁇ l reverse transcription mixed primers per well.
  • the sequence of the bridging primer is 5'-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3', and the 3' end is modified with ddC to prevent the extension of the bridging primer during reverse transcription, resulting in the generation of by-products.
  • reverse transcription primers reverse transcription sequences
  • the core sequence is 6 ⁇ N.
  • Each primer is placed independently in each well. This 6 ⁇ N random sequence can be used as a part of the cell label sequence.
  • Subsequent combination with the cell label sequence on the molecularly labeled magnetic beads in Example 1 is used to identify the cells from which the mRNA corresponding to each sequence in the subsequently constructed sequencing library is taken, so that there are a total of 96 ⁇ 96 ⁇ 96 ⁇ 96 single-stranded oligonuclei Nucleotides are used to identify cells and are sufficient for one-time use of millions of cells.
  • the 5' end of the molecular tag sequence of the last segment of the reverse transcription primer is modified by phosphorylation to provide a phosphate group for the ligation reaction.
  • n represents any one of A/T/C/G, which is randomly synthesized.
  • V at the 3' end means any of A/C/G, N means any of A/T/C/G, randomly synthesized, the main purpose is to make the primer bind to the end of the polyA tail and avoid binding to The middle part of the polyA tail, the specific sequence is as follows: 5'-[phos]ACACTCTTTCCCTACACGACGNNNNNNNnnnnnnnnnnnnTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN
  • Configure 310 ⁇ l reverse transcription system (50 ⁇ l dNTP, 200 ⁇ l buffer, 50 ⁇ l reverse transcriptase, 10 ⁇ l RNase inhibitor), mix well, and dispense into 96-well plates containing reverse transcription mixed primers, 3.1 ⁇ l per well. After the fixation, the two kinds of cells were centrifuged and washed once at a speed of 500 g, and 2.5 million cells were taken for mixing.
  • the cells were resuspended in 500 ⁇ L DPBS solution, and then the cell suspension was dropped into the microwell plate, so that more than 80% of the microwells were covered with cells ( FIG. 3 ).
  • lysis solution (ddH 2 O, 10% SDS, 50% formamide (vol/vol) and 3 ⁇ SSC) dropwise to the microwell plate covered with magnetic beads, and incubate for 30 minutes at room temperature to make the magnetic beads
  • the bridging sequence and the bridging primer on the cell are sufficiently complementary to hybridize.
  • place the microwell plate upside down on the magnet collect the magnetic beads with molecular marker-mRNA complexes, transfer them to a 1.5ml EP tube, wash twice, suck up the residual liquid with a 20 ⁇ l pipette gun, and pour it into the container.
  • the molecularly labeled magnetic beads were washed three times on the magnetic stand, the supernatant was discarded, and the PCR reagent mixture (KAPA HiFi Hot Start Ready Mix 1 ⁇ , TSO-PCR primer 0.1 ⁇ M) was added.
  • the TSO-PCR primer sequence is: 5'-AAGCAGTGGTATCAACGCAGAGT-3'.
  • PCR program pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 20 seconds, annealing at 67°C for 15 seconds, extension at 72°C for 6 minutes, repeated 12 times; extension at 72°C for 5 minutes, and incubation at 4°C to obtain a large amount of labeled cDNA.
  • PCR products were purified using Novizyme purification beads. Shake and mix the purified magnetic beads at room temperature for at least 30 minutes before use. The purification steps are as follows:
  • the following cDNA sequencing library construction method was used to construct the gene sequencing library. After the gene sequencing library was constructed, it was sent to Ringpu for sequencing. The data returned by the sequencing were split, screened and compared to obtain the gene expression profile. By importing this matrix file into R language analysis, the matrix data can be converted into a visualized graph. It can be seen from Figure 6 that there is a very small amount of double-cell contamination, which can reach the level of single-cell high-throughput sequencing.
  • Vazyme TD512 kit was used.
  • reaction component in sequence to a sterilized PCR tube: 5 ⁇ TTBL, 4 ⁇ l; DNA, 5 ng; TTE Mix V1, 5 ⁇ l; ddH 2 O to make up to 20 ⁇ l.
  • the kit used is: TruePrep TM Index Kit V2 for (Vazyme#TD202), the specific experimental steps refer to the instructions of the kit.
  • the initial PCR product volume should be 50 ⁇ l. Because the sample volatilization during the PCR process will cause the volume of the product to be less than 50 ⁇ l, you must use sterilized distilled water to make up the volume to 50 ⁇ l before performing the following operations, otherwise the sorting length will be inconsistent with the expectation.
  • the amount of magnetic beads used in the two rounds is as follows:
  • the amount of AMPure XP magnetic beads used in the first round R1 30.0 ⁇ l (0.60 ⁇ )
  • the amount of AMPure XP magnetic beads used in the second round R2 7.5 ⁇ l (0.15 ⁇ ).
  • step 5 Repeat step 5 for a total of two rinses.
  • Example 2 Same as in Example 2, after the cells with molecular labels were dropped off the plate, DPBS solution was used to wash off excess molecularly labeled magnetic beads, and three different formulations of lysates were added to three microwell plates, followed by the control lysate ( 0.1M Tris-HCl pH 7.5, 0.5M LiCl, 1% SDS, 10mM EDTA and 5mM dithiothreitol), Lysis Solution 20 (ddH 2 O, 1% SDS, 20% Formamide and 3 ⁇ SSC), Lysis Solution 50 (ddH 2 O, 1% SDS, 50% formamide and 3 ⁇ SSC), fully lysed and incubated for 30 minutes, and the subsequent steps were the same as in Examples 2 and 3.
  • control lysate 0.1M Tris-HCl pH 7.5, 0.5M LiCl, 1% SDS, 10mM EDTA and 5mM dithiothreitol
  • Lysis Solution 20 ddH 2 O, 1% S
  • SDS plays a major role in cleavage
  • formamide promotes the hybridization of nucleic acid molecules
  • SSC assists in improving hybridization efficiency.
  • the gene sequencing library was constructed and sent to Ringpu for sequencing, using the HiSeq2500PE125 sequencing strategy. Analysis of the sequencing results found that lysate 50 significantly increased the number of cells with UMI greater than 500 and the average number of genes (Figure 7), which greatly improved the reaction efficiency of the platform.
  • FIG. 8 shows the results of tSNE analysis of mouse testis cells, showing that mouse testis can be divided into 14 subgroups.
  • the present invention can also be applied to single-cell sequencing platforms based on microfluidics. After the fixed cells with a round of cell labeling were obtained by the method described in Example 2, the reaction was carried out using 10 ⁇ Chromium Chip E (10 ⁇ Genomics #2000121 ) from the company.
  • Inlet 1 inject 75 ⁇ l fixed cells mixed with ligation system (10 ⁇ l cells, 50.5 ⁇ l enzyme-free water, 7.5 ⁇ l T4 ligation buffer, 3 ⁇ l T4 ligase, 1.5 ⁇ l 10 ⁇ Reducing Agent B and 2.5 ⁇ l 100mM bridging primer), import 2 Inject 40 ⁇ l of single-cell ATAC gel microbeads containing lysate (10 ⁇ Genomics #2000132, the bead sequence is as described in Example 1), and inject 240 ⁇ l of Partitioning Oil (10 ⁇ Genomics #220088) into inlet 3. Subsequently, the emulsion-coated microbeads were incubated at 37°C for 1.5 hours to make the ligation reaction sufficient. After the reaction, the microbeads were collected for excision, single-strand synthesis, and double-strand synthesis reactions according to the protocol described in Example 2, and finally a cDNA library was obtained by PCR amplification and purification.
  • ligation system 10 ⁇ l
  • Anneal the labeled magnetic beads and bridging primers in Example 1. Mix the magnetic beads with the bridging primer (sequence of the bridging primer: 5'-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA-3'), and gradient anneal to form sticky ends. Sequences not bound by the bridging primer were excised using exonuclease EXO I. After excision is complete, wash once with 150 ⁇ l of TE-SDS and TE-TW, and finally resuspend with TE-TW, and store at 4°C for future use.
  • Tn5 transposase naked enzyme was purchased from Nanjing Novizan Biotechnology Co., Ltd.
  • the transposase and embedding buffer were provided by (Vazyme) Tn5 Transposome (S111) kit produced by Nanjing Novizan Biotechnology Co., Ltd.
  • the specific molecular tag 10 ⁇ N contained in the specific tag fragment is the core sequence of the cell tag sequence, and the core sequence corresponding to each Tn5 complex is different. choices, so there are 4 10 choices for a 10 ⁇ N sequence.
  • N represents any one of A/T/C/G, which is randomly synthesized.
  • RSBT buffer ddH 2 O, 10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl 2 , 1% BSA, 0.1% Tween-20
  • Nuclei were fixed with 1% formaldehyde for 10 minutes at room temperature. Fixed crosslinks were terminated with glycine.
  • Mouse fibroblasts (3T3) and human embryonic kidney cells (293T) were mixed with 2 million each, washed with PBS, transferred to 1.5ml EP tube, and then lysed with 1ml lysate (ddH 2 O, 10mM Tris-HCl pH7 .4, 10 mM NaCl, 3 mM MgCl 2 , 1% BSA, 0.1% Tween-20, 0.1% IGEPAL CA-630, 0.01% digitonin) resuspended, and lysed on ice for 3 min.
  • 1ml lysate ddH 2 O, 10mM Tris-HCl pH7 .4, 10 mM NaCl, 3 mM MgCl 2 , 1% BSA, 0.1% Tween-20, 0.1% IGEPAL CA-630, 0.01% digitonin
  • RSBT buffer 1ml RSBT buffer to suspend the nuclei and count them for later use after filtration.
  • the sample was taken out, thawed in an oven at 37°C for 2 minutes, centrifuged at 500g/5min, the supernatant was discarded, resuspended with 200 ⁇ l lysate, and placed on ice for 3 minutes. Wash and filter with RSBT and count for later use.
  • the transposase embedding complex in Example 3 was added to a 96-well plate containing cell nuclei, 2.5 ⁇ l per well, and placed at 55° C. for 30 minutes to react. After the reaction, 25 ⁇ l of 2 ⁇ stop solution (25 ml of 40 mM EDTA, 3.9 ⁇ l of 6.4M Spermidine) was added to each well, and left standing at 37° C. for 15 minutes. Collect the liquid and mix it into a 15ml centrifuge tube, 500g/5min, discard the supernatant, wash once with 1ml RSBT.
  • 2 ⁇ stop solution 25 ml of 40 mM EDTA, 3.9 ⁇ l of 6.4M Spermidine
  • Tn5-labeled nuclei Drop the cell nucleus suspension into the microwell plate, use a centrifuge to briefly centrifuge to make 80-90% of the nucleus fall into the microwell, capture 0-10 nuclei in each well, and pass the cell label on Tn5 4 pairs in the same well Cellular DNA fragments are distinguished. Anneal the bridging primer to the labeled beads so that the fragments on the labeled beads form cohesive ends. Add 200,000 molecularly labeled magnetic beads to the microwell plate with cell nuclei, place on a magnet and mix gently so that the magnetic beads cover more than 99% of the microwells, and use RSBT solution to wash away excess molecularly labeled magnetic beads.
  • N represents any one of A/T/C/G, which is randomly synthesized.
  • S represents the thio modification of the terminal base to improve the stability of the terminal base.
  • the ultra-high-throughput single-cell sequencing with chromatin transposase accessibility of the present invention can also be applied to a single-cell sequencing platform based on microfluidics. After obtaining fixed cell nuclei with a round of cell labeling by the method described in Example 6, the reaction was performed using 10 ⁇ Chromium Chip E (10 ⁇ Genomics #2000121 ) from the company.
  • Inlet 1 inject 75 ⁇ l fixed cells mixed with ligation system (10 ⁇ l cells, 50.5 ⁇ l enzyme-free water, 7.5 ⁇ l T4 ligation buffer, 3 ⁇ l T4 ligase, 1.5 ⁇ l 10 ⁇ Reducing Agent B and 2.5 ⁇ l 100mM bridging primer), import 2 Inject 40 ⁇ l of single-cell ATAC gel microbeads containing lysate (10 ⁇ Genomics #2000132, the bead sequence is as described in Example 1), and inject 240 ⁇ l of Partitioning Oil (10 ⁇ Genomics #220088) into inlet 3. Subsequently, the emulsion-coated microbeads were incubated at 37°C for 1.5 hours to make the ligation reaction sufficient.
  • ligation system 10 ⁇ l cells, 50.5 ⁇ l enzyme-free water, 7.5 ⁇ l T4 ligation buffer, 3 ⁇ l T4 ligase, 1.5 ⁇ l 10 ⁇ Reducing Agent B and 2.5 ⁇ l 100mM bridging
  • Example 7 After the reaction, microbeads were collected to carry out excision, single-strand synthesis, and double-strand synthesis reactions according to the protocol described in Example 7, and finally a gene sequencing library was obtained by PCR amplification and purification, sequencing and bioinformatics analysis.
  • the gene sequencing library was sequenced and bioinformatically analyzed according to the procedure in Example 7.
  • the ultra-high-throughput single-cell sequencing of the chromatin transposase accessibility of the present invention can also be applied to the Tn5 embedding method that both ends are marked.
  • each well of 96 wells contains one embedded fragment, which is a specific tag fragment + Mosaic Ends fragment specific between wells, and the specific tag fragment + Mosaic Ends fragment is annealed to obtain a specific Sex molecular tag sequence, that is, the bridge sequence 2-cell tag 4-Mosaic Ends/Mosaic Ends in Figure 9.
  • the cell nuclei were labeled according to the protocol in Example 6, and the labeled cell nuclei were captured in a microwell plate, ligated, and extended.
  • P7 adapter-Mosaic Ends primer ie, a primer including the sequence of the P7 adapter and Mosaic Ends fragment
  • the upper list chain products are collected by high temperature denaturation, and the magnetic beads are removed.
  • Use index P5 and index P7 primers to PCR amplify and purify to obtain a gene sequencing library (as shown in Figure 18).
  • the gene sequencing library was sequenced and bioinformatically analyzed according to the procedure in Example 7.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了一种超高通量单细胞测序方法，本发明超高通量单细胞测序方法先使用反转录序列对细胞内进行一次细胞内反转录，或者先使用特异性分子标签转座酶包埋复合物进行细胞核内染色质转座酶可及性基因组序列的转座，再将细胞或细胞核通过微孔板技术或微流控技术使一个分子标记微珠与一个或多个细胞处于分隔的空间中，并在裂解液作用下裂解细胞/细胞核，在桥连引物的帮助下将序列与分子标记微珠上的分子标记序列连接，并通过PCR扩增获得大量序列，构建获得cDNA测序文库，然后进行高通量测序，一次测序便可以获得上百万个单细胞的特异性转录组/基因组开放性信息。大大提高了单细胞测序的通量。

Description

一种超高通量单细胞测序方法 技术领域
本发明涉及单细胞测序技术领域，特别是涉及一种超高通量单细胞测序方法。
背景技术
自2009年汤富酬提出单细胞测序技术以来，单细胞高通量测序平台就如雨后春笋般不断涌现，例如以微流控为主的Drop-seq和inDrop-seq平台，以微孔板为主的Microwell-seq和Seq-well平台以及常见的商用平台10×Gennomics。然而，以微孔板为例，当细胞投入量达到一定程度时，容易出现一个微孔中含有两个甚至更多细胞的情况出现，导致细胞与细胞间转录本的污染。为了避免这种现象的产生，通过分析发现当微孔板中细胞的落孔率约为微孔板总孔数的1/10时，可实现每个微珠仅捕获一个细胞。但这样会导致大量的液滴或者微孔仅仅只有空微珠而没有细胞，大大降低了实验的效率，增加了实验成本，导致这些平台单次实验的细胞通量只能以万为单位。然而，以小鼠和人为例，单个物种个体的细胞总量超过万亿，当下平台通量远远达不到测序的要求，测序通量的不足容易丢失大量的生物信息，因此提高单次测序的通量显得尤为重要。
近年来以单个细胞为独立反应体系的超高通量技术也在不断发展，例如sci-RNA-seq和SPLiT-seq，他们首先将新鲜细胞固定打孔，利用反转录给每一个细胞带上一轮独有的分子标记，随后利用split-pool再给细胞带上不同的分子标记，最终通过分子标记的多重组合使得每一个细胞中的转录本带上独有的分子标记，大大提升了单次实验的通量。但由于这些方法是基于细胞体内的连接反应，存在反应效率低、细胞与细胞之间的转录本易泄露导致污染率较高等问题，降低了方法的实用性。
2019年12月奥地利科学院分子医学研究中心的Paul等(Datlinger,P.,et al.,Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing.BioRxiv,2019.)利用反转录给固定的细胞带上一轮含有96/384种的分子标记后，结合微流控的方法将单细胞测序的通量提高了15倍。但以微流控为基础的测序平台细胞并不是平行进行实验的，批次效应问题比较显著，UMI/Gene比值异常，且存在设备昂贵、不易携带、测序成本高等缺点。
在基因组中，大部分的染色质紧密缠绕在细胞核内，不具有转录活性。染色质状态以细胞类型特定的方式进行动态调节，在特定细胞状态下部分致密的染色质变得松散，这些松散的染色质被称为开放染色质(open chromatin)或可接近性染色质(accessible chromatin)。检测细胞染色质的开放情况，可以得到细胞的转录调控信息，比如转录因子在什么位置能够与基因启动子结合、细胞的哪些基因可能被高效转录等。 常用的检测手段是ATAC-seq、DNase-seq、MNase-seq、FAIRE-seq和ChIP-seq等，这些方法基于不同的原理对染色质开放区域进行打断和标记，其中ATAC-seq(高通量染色质转座酶可及性测序方法，assay for transposase-accessible chromatin with high-throughput sequencing)使用修饰的Tn5转座酶，可以将特定DNA序列作为转座子随机插入到染色质的开放区域，能够完整地将整个开放区域的序列直接捕获下来，所以ATAC-seq现在被广泛应用到开放染色体的测序当中。
发明内容
本发明提供了一种超高通量单细胞测序方法，该单细胞测序方法能够一次性获得上百万个单细胞的特异性转录组信息。
本发明首先提供了一种超高通量单细胞测序方法，包括以下步骤：
(1)准备如下试剂：
a)分子标记微珠，所述分子标记微珠包括微珠本体和耦联的分子标记序列，该分子标记序列包括顺序排列的：
通用引物序列，作为PCR扩增时的引物结合区域；
第一细胞标签序列；
第一搭桥序列；
b)反转录序列，用于细胞内反转录，所述反转录序列包括顺序排列的：
第二搭桥序列；
第二细胞标签序列，与第一细胞标签序列配合组成细胞标签序列，所述细胞标签序列用于标识所构建测序文库中各序列所对应的mRNA取自的细胞；
分子标签序列，用于标识所构建测序文库中各序列所对应的mRNA；
多聚T尾，用于与细胞中带有poly-A序列的mRNA互补配对；
c)桥连引物，用于将上述a)中的标记序列与b)中的反转录序列进行连接，所述桥连引物两端具有分别与所述第一搭桥序列和第二搭桥序列互补配对的序列；
(2)取待测序的细胞样品，加入反转录序列进行细胞内反转录，使反转录序列的多聚T尾端连上将细胞内mRNA序列反转录后得到的cDNA序列，获得反转录序列-cDNA序列；
(3)将步骤(2)经细胞内反转录后的细胞通过微孔板技术或微流控技术使一个分子标记微珠与一个或多个细胞处于分隔的空间中，并在裂解液作用下裂解细胞，孵育，通过桥连引物分别与所述第一搭桥序列和第二搭桥序列互补配对，然后使用连接酶连接，使第一搭桥序列和第二搭桥序列连接获得与微珠耦联的分子标记序列-反转录序列-cDNA序列；
(4)收集耦联有分子标记序列-反转录序列-cDNA序列的微珠，进行PCR扩增获得带有第一细胞标签序列、第二细胞标签序列和分子标签序列的cDNA序列；
(5)将步骤(4)获得的产物构建cDNA测序文库，然后进行高通量测序，获得上百万个单细胞的特异性转录组信息。
该测序方法为一种转录组测序方法。通过将细胞标签序列分为第一细胞标签序列和第二细胞标签序列，第二细胞标签序列以反转录序列的形式在细胞内反转录时在各mRNA的反转录序列中引入，从而在出现一个分子标记微珠结合了多个细胞的情况下，能够通过这段第二细胞标签序列来区分开，否则，仅依靠分子标记微珠上的第一细胞标签序列，就无法区分结合同一分子标记微珠的多个细胞来源的序列。现有技术中，为了避免一个分子标记微珠结合多个细胞，需要严格控制条件，比如，将实验的微孔板上的微孔尽量制备成只容纳一个分子标记微珠和一个细胞的大小(这种情况下，分子标记微珠和细胞的相对大小不宜过大，不然，不容易实现一个分子标记微珠结合一个细胞)，同时需要控制细胞的落孔率在较低水平，以便细胞足够分散开，但还是无法避免出现一个分子标记微珠结合多个细胞的情况，而最终测序结果会将这种情况下的序列误判为来自同一个细胞。
同时，第二细胞标签序列与第一细胞标签序列配合组成细胞标签序列，增加了细胞标签序列的组合数量，可以一次实现更大量细胞的检测。
本发明又提供了一种超高通量单细胞测序方法，包括以下步骤：
(1)准备如下试剂：
a)分子标记微珠，所述分子标记微珠包括微珠本体和耦联的分子标记序列，该分子标记序列包括顺序排列的：
通用引物序列，作为PCR扩增时的引物结合区域；
第一细胞标签序列；
第一搭桥序列；
b)特异性分子标签转座酶包埋复合物，包括Tn5转座酶和特异性分子标签序列，其中特异性分子标签序列包括顺序排列的：
第二搭桥序列；
第二细胞标签序列，与第一细胞标签序列配合组成细胞标签序列，所述细胞标签序列用于标识所构建测序文库中各序列来源的细胞；
Mosaic Ends序列，用于与Tn5转座酶结合，所述Mosaic Ends序列为双链结构，其中一条链与第二细胞标签序列连接；
c)桥连引物，用于将上述a)中的分子标记序列与b)中的特异性分子标签序列进行连接，所述桥连引物两端具有分别与所述第一搭桥序列和第二搭桥序列互补配对的序列；
(2)取待测序的细胞样品，提取细胞核；
(3)向步骤(2)提取的细胞核中加入特异性分子标签转座酶包埋复合物进行转 座反应；
(4)将经过转座反应后的细胞核通过微孔板技术或微流控技术使一个分子标记微珠与一个或多个细胞核处于分隔的空间中，并在裂解液作用下裂解细胞核，孵育，通过桥连引物分别与所述第一搭桥序列和第二搭桥序列互补配对，然后使用连接酶连接，使第一搭桥序列和第二搭桥序列连接获得与微珠耦联的分子标记序列-特异性分子标签序列-染色质转座酶可及性基因组序列；
(5)收集耦联有分子标记序列-特异性分子标签序列-染色质转座酶可及性基因组序列的微珠，进行PCR扩增获得带有第一细胞标签序列、第二细胞标签序列和特异性分子标签序列的染色质转座酶可及性基因组序列；
(6)将步骤(5)获得的产物构建染色质可及性测序文库，然后进行高通量测序，获得上百万个单细胞的染色质转座酶可及性基因组序列信息。
该测序方法为一种染色质转座酶可及性测序方法。通过将细胞标签序列分为第一细胞标签序列和第二细胞标签序列，第二细胞标签序列在Tn5转座酶转座反应时带入到基因组相应的序列上，从而在出现一个分子标记微珠结合了多个细胞的情况下，能够通过这段第二细胞标签序列来区分开，否则，仅依靠分子标记微珠上的第一细胞标签序列，就无法区分结合同一分子标记微珠的多个细胞来源的序列。
优选的，所述微珠与分子标记序列耦联方式为：分子标记序列5’端的核苷酸的C6位上使用胺基取代羟基，微珠表面修饰有羧基，通过羧基与氨基缩合耦联。由于分子标记序列为单链寡核苷酸，其5’端的第一个核苷酸上的羟基用胺基取代，微珠表面修饰羧基，通过氨基与羧基的反应，将分子标记序列耦联到微珠上。
优选的，所述分子标签序列至少部分为随机合成的随机序列。
优选的，所述第一细胞标签序列包括多个特异性片段，所述第二细胞标签序列包括至少一个特异性片段，不同位置的特异性片段选自相同的或不同的特异性片段库，所述第一细胞标签序列和第二细胞标签序列利用所述特异性片段的排列组合方式不同标识细胞。
更优选的，所述分子标记微珠的制备方法包括以下步骤：
(1)用于合成分子标记序列的引物根据特异性片段的数量分为多条引物，每条引物包含一条特异性片段，各引物之间具有用于搭桥连接、互补的接头序列，其中对应于分子标记序列5’端的那条引物还包括所述通用引物序列，对应于分子标记序列3’端的那条引物还包括所述第一搭桥序列；
(2)将对应于分子标记序列5’端的那条引物与微珠本体耦联，然后通过PCR方法将剩余引物依次退火、延伸，从5’端到3’依次串接分子标记序列的其余特异性片段，制备得到所述分子标记微珠。
优选的，所述分子标记序列为：5’-TTTAGGGATAACAGGGTAATAAGCAGTGG  TATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAGCGTC-3’，其中，N表示A/T/C/G中的任一种，为随机合成。所述反转录序列为：5’-[phos]ACACTCTTTCCCTACACGACGNNNNNN nnnnnnnnnnTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’，其中，5’端加上磷酸化修饰为连接反应提供磷酸基团；N表示A/T/C/G中的任一种，为随机合成；n表示A/T/C/G中的任一种，为随机合成；3’端V表示A/C/G中的任一种，V为随机合成。所述特异性分子标签序列为：5’-ACACTCTTTCCCTACACGACGNNNNNNNNNNAGATGTGTA TAAGAGACAG-3’，其中，N表示A/T/C/G中的任一种，为随机合成，互补形成双链的Mosaic Ends序列为：5’-CTGTCTCTTATACACATCT-3’。所述桥连引物为：5’-CGT CGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3’，ddC为双脱氧胞苷修饰。或者3’端的ddC去掉。反转录序列中，6×N的随机序列作为第二细胞标签序列，10×n的随机序列作为分子标签序列。
对于染色质转座酶可及性测序来说，所述特异性分子标签转座酶包埋复合物中一个转座酶复合物携带两个基因片段，两个基因片段均为所述特异性分子标签序列，或者一个基因片段为所述特异性分子标签序列，另一个基因片段为通用型序列，通用型序列包括：
扩增用引物结合序列，作为PCR扩增时的引物结合区域；
Mosaic Ends序列，用于与Tn5转座酶结合，所述Mosaic Ends序列为双链结构，其中一条链与扩增用引物结合序列连接。
优选的，待测序的细胞样品中含有2种或2种以上的细胞。本申请超高通量单细胞测序方法可以实现多种细胞的同时测序。
优选的，所述微珠本体为磁珠；将经细胞内反转录后的细胞(反转录测序)或经过转座反应后的细胞核(染色质转座酶可及性测序)加入到微孔板中，然后加入分子标记微珠，微孔板中的微孔直径大小为刚好容纳一个分子标记微珠和一个或多个细胞/细胞核；细胞或细胞核加入微孔板中的落孔率控制在大于80％；分子标记微珠加入微孔板中的落孔率大于99％。
由于本申请超高通量单细胞测序方法可以实现一个分子标记微珠结合多个细胞，所以在微珠本体为磁珠、用微孔板方法时，可以实现细胞加入微孔板时落孔率的大大提高。
本发明除了在微珠本体为磁珠、用微孔板方法的单细胞测序之外，也可以用于微流控方法的单细胞测序平台。
更优选的，所述微孔板中微孔深度30-160μm、微孔直径20-150μm；所述微珠本体的直径为20-145μm。
更优选的，所述微孔板的制备方法为：(1)在硅片上刻蚀出微孔作为初始模具； (2)在初始模具上浇注聚二甲基硅氧烷，成型后取下聚二甲基硅氧烷成为具有微柱的第二次模具；(3)在第二次模具上浇注热熔的质量体积比为4％～6％的琼脂糖，冷却成型后，取下琼脂糖即为所述微孔板。
本发明超高通量单细胞测序方法能够进行单细胞的超高通量测序，一次测序的细胞数量可以达到上百万个单细胞，大大提高了单细胞测序的通量。
附图说明
图1为蜂窝排列微孔板示意图。
图2为分子标记磁珠制备流程图。
图3为细胞落入微孔板示意图。
图4为构建cDNA文库流程图，其中通用序列包括图2所述通用引物序列，细胞标签序列1，接头序列1，细胞标签序列2，接头序列2，细胞标签序列3。
图5为制备的cDNA测序文库片段大小分布图。
图6为人鼠混合细胞分群对比图。
图7为不同裂解液测序读长/基因对比图。
图8为小鼠睾丸细胞tSNE分析结果图。
图9为接头序列与Tn5转座酶进行接头包埋，其中两种接头序列分别包含特异性分子标签序列和通用型序列，接头序列通过与Tn5裸酶孵育产生Tn5包埋复合物，单个Tn5包埋复合物同时携带特异性分子标签序列和通用型序列。
图10为构建ATAC文库流程图，其中，通用序列包括图1中的通用引物序列、细胞标签序列1、接头序列1、细胞标签序列2、接头序列2和细胞标签序列3；搭桥序列包括搭桥序列1和搭桥序列2。
图11为制备的基因测序文库片段大小分布图。
图12为人鼠混合细胞分群对比图。
图13为人鼠样品DNA片段大小分布图。
图14为人鼠样品peak注释分布图。
图15为小鼠TSS富集图。
图16为小鼠单细胞ATAC和群体ATAC peak重叠分布图。
图17为小鼠单细胞ATAC和群体ATAC read在8号染色体0-25Mbp分布比较图。
图18为双端都标记的Tn5包埋方法构建ATAC文库流程图，其中，通用序列包括图1中的通用引物序列、细胞标签序列1、接头序列1、细胞标签序列2、接头序列2和细胞标签序列3。
具体实施方式
实施例1
1、微孔板制备
根据实验规模设计微孔板大小(比如，人293T细胞和鼠3T3细胞各50万个，孔板大小为1.8cm×1.8cm)，并在硅片上蚀刻出微孔作为初始模具，微孔为圆柱体型，其中微孔深度60μm、微孔直径50μm、孔间距70μm。接下来在硅片上浇注聚二甲基硅氧烷(PDMS)，成型后拿下PDMS成为板上有微柱的第二次模具，最终实验使用的微孔板是浓度为5％(质量比)的琼脂糖(用无酶水配制)，热融后浇注在PDMS微柱板上冷凝成型，此时的琼脂糖板揭下来后就是具有一定厚度的微孔板(图1)。保存时加上对细胞无害的DPBS-EDTA混合液，加盖保存在4℃冰箱中，即做即用能保证微孔板良好的工作状态。
2、分子标记磁珠制备
磁珠购自苏州知益微球科技有限公司(货号MagCOOH-20190725)，表面羧基包被，直径45μm。分子标记磁珠制备过程如图2所示，共4个步骤：
(1)设计分子标记序列，将分子标记序列分成三段，相邻两段之间设置有用于将相邻两段通过PCR连接起来的接头序列，其中5’开始的第一段包括通用引物序列和部分细胞标签序列，最后一段含有部分细胞标签序列及整个分子标签序列、桥连互补序列，除第一段外，其余序列均为相应序列的互补序列。
(2)各段序列如下表1所示。
表1
Figure PCTCN2021119166-appb-000001
其中，6×N为细胞标签序列的核心序列，对应每个磁珠的这个核心序列均不同，且对应同一磁珠的三段序列中的6×N序列也不同，由于每个位点有A/T/C/G这4种选择，所以6×N的序列可以有4 6种选择。N表示A/T/C/G中的任一种，为随机合成。
(3)分别合成所有序列，其中所有序列中属于细胞标签序列部分均设计96种序列，每种独立放置，第一段序列5’端的核苷酸的C6位上使用胺基取代羟基。
(4)将等量的磁珠分别与96种第一段序列偶联，然后收集获得96种带修饰的磁珠，混合均匀后，再均分为96等分，与96种第二段序列混合后进行PCR序列延伸，然后再均分为96等分，与96种第三段序列混合后进行PCR序列延伸，然后变性解链获得具有96×96×96种单链寡核苷酸修饰的磁珠。
完成后，分子标记的序列如下：5’-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAGCGTC-3’，可用于超高通量单细胞测序。
实施例2
特异性转录组超高通量单细胞测序。
1、人293T、鼠3T3混合细胞测试。
小鼠胚胎干细胞(ESC)3T3和人胚肾细胞(293T)各500万分别缓慢滴加5-10ml甲醇(-20℃预冷)在-20℃中固定30分钟，同时，将搭桥引物分装至八联管中，每孔6.5μl，再分装至含有0.5μl反转录引物的96孔板中，静置混匀形成每孔共1μl反转录混合引物。桥连引物序列为5’-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3’，3’端加上ddC修饰防止搭桥引物反转录时进行延伸导致副产物的产生。
反转录引物(反转录序列)同上述细胞标签序列也为96种，核心序列为6×N，每种引物每孔独立放置，这段6×N随机序列可以作为细胞标签序列的一部分，后续与实施例1中分子标记磁珠上的细胞标签序列组合用于标识后续所构建测序文库中各序列所对应的mRNA取自的细胞，从而共有96×96×96×96种单链寡核苷酸用来标识细胞，足够用于一次性百万级细胞的使用。反转录引物的最后一段的分子标签序列5’端加上磷酸化修饰为连接反应提供磷酸基团，10×n中n表示A/T/C/G中的任一种，为随机合成，3’端V表示A/C/G中的任一种，N表示A/T/C/G中的任一种，随机合成，主要目的是为了使引物结合到polyA尾的末端，避免结合到polyA尾的中间部分，具体的序列如下：5’-[phos]ACACTCTTTCCCTACACGACGNNNNNNnnnnnnnnnnTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’。
配置310μl反转录体系(50μl dNTP，200μl缓冲液，50μl反转录酶，10μl RNA酶抑制剂)，混匀，分装至含有反转录混合引物的96孔板中，每孔3.1μl。随后将固定完成后的两种细胞在500g转速下离心清洗一次，各取250万进行混合。将混匀后的细胞(每孔约5万)平均分装至含有预混反转录体系(6μl细胞悬液，0.5μl dNTP，1μl反转录混合引物，2μl缓冲液，0.5μl反转录酶，0.1μl RNA酶抑制剂)的96孔板中42℃反应1.5小时。反转录结束后，将96孔板中的细胞先利用排枪收集至八联管中，再集中转移至干净的1.5ml EP管中，用DPBS溶液(Gibco公司，货号14190-144)清洗一次，500g离心5分钟。吸取上清液后，将细胞重悬于500μL DPBS溶液中，再将细胞悬液滴加入微孔板中，使大于80％的微孔中落有细胞(图3)。向落有细胞的微孔板中加入20万个分子标记磁珠，置于磁铁上轻轻混匀，使磁珠覆盖99％以上的微孔，使用DPBS溶液洗去多余的分子标记磁珠。向铺满磁珠的微孔板中缓慢滴加200μL裂解液(ddH 2O，10％SDS，50％甲酰胺(vol/vol)和3×SSC)，室温裂解孵育30分钟，使磁珠上的搭桥序列和细胞上的搭桥引物充分互补杂交。孵育完成后，将微孔板倒置于磁铁上，收集带有分子标记-mRNA复合物的磁珠，转移至1.5ml EP管中，清洗两次，用20μl移液枪吸取残留的液体，向装有分子标记磁珠的EP管中加入连接混合液50μL(T4连接酶2μl，T4缓冲液5μL，RNase抑制剂1μL，dNTP 2μL，30％PEG8000 5μL，ddH 2O 35μL)，置于37℃反应1小时。
连接反应完成后，把分子标记磁珠在磁力架上清洗三次，吸弃上清，加入外切酶EXON Ⅰ混合液200μL(EXON Ⅰ缓冲液1×，EXON Ⅰ 1×)37℃反应0.5小时，清除磁珠上没有捕获mRNA的单链核苷酸序列。外切结束后，把分子标记磁珠在磁力架上清洗三次，吸弃上清，加入500μL 0.1％的NaOH溶液处理5分钟，获得单链化cDNA，以便于后续二链合成反应。把NaOH处理过的分子标记磁珠在磁力架上清洗三次，清除残留的NaOH，随后加入二链合成混合液100μL(20μL反转录缓冲液，40μL 30％PEG8000，10μL 10mM dNTP，10μL 100μM随机引物，2.5μL Klenow聚合酶和17.5μL ddH 2O)于37℃反应1小时，其中随机引物序列为5’-AAGCAGTGGTATCAACGCAGAGTGANNNGGNNNB-3’，B表示G/T/C中的一种，N表示A/T/C/G中的任一种，随机合成。随机引物序列将随机结合在NaOH处理的磁珠单链序列上，聚合酶沿该引物3’方向继续合成互补链，其中NNNGGNNNB为随机结合序列。
二链合成反应结束后，把分子标记磁珠在磁力架上清洗三次，吸弃上清，加入PCR试剂混合液(KAPA HiFi Hot Start Ready Mix 1×，TSO-PCR引物0.1μM)。其中，TSO-PCR引物序列为：5’-AAGCAGTGGTATCAACGCAGAGT-3’。PCR程序：98℃预变性3分钟；98℃变性20秒，67℃退火15秒，72℃延伸6分钟，重复12次；72℃延伸5分钟，4℃保温，即得到大量标记cDNA。使用诺唯赞纯化磁珠纯化PCR产物。使用前将纯化磁珠震荡混匀并置于室温至少30分钟，纯化步骤如下：
(1)加50μL纯化磁珠到上述PCR反应体系中，使用移液器混匀10次以上保证整个体系均匀；(2)室温孵育10分钟；(3)将PCR管置于磁力架上5分钟保证纯化磁珠完全吸附；(4)保持PCR管在磁力架上，小心弃去上清；(5)加入200μL新鲜配制的80％乙醇，孵育30秒后弃掉上清；(6)重复上述步骤一次；(7)开盖，空气中干燥8分钟；(8)加入13μL洗脱缓冲液(Elution buffer)到PCR管中覆盖纯化磁珠，将PCR管从磁力架上取下并重悬纯化磁珠；(9)室温孵育2分钟，吸取12μL为最终的cDNA文库(上述反应具体流程见图4)；(10)利用Agilent 2100 Bioanalyzer对cDNA文库片段大小进行分析(图5)，得到的cDNA文库片段在300-1000bp。
以下述cDNA测序文库构建方法构建基因测序文库，基因测序文库构建好后送至瑞普公司进行测序。测序返回的数据经过拆分筛选比对，得到基因表达谱。将这个矩阵文件导入R语言分析，就能将矩阵数据转换为可视化的图。由图6可知，存在极少量双细胞污染，可以达到单细胞高通量测序水平。
2、cDNA测序文库构建。
(1)5ng起始DNA片段化
使用Vazyme公司TD512试剂盒。
(a)于室温解冻5×TTBL(TruePrep Tagment Buffer L)，上下颠倒混匀后备用。确认5×TS(Terminate Solution，反应终止液)处于室温，并轻弹管壁确认有无沉淀。如有沉淀，可于37℃加热并剧烈震荡充分混匀沉淀即会溶解。
(b)在灭菌PCR管中依次添加各反应组分：5×TTBL，4μl；DNA，5ng；TTE Mix V1，5μl；ddH 2O补足至20μl。
(c)使用移液器轻轻吹打20次充分混匀。
(d)将PCR管置于PCR仪中，设置如下反应程序：55℃10min；10℃保温。
(e)立即向反应产物中加入5μl 5×TS，使用移液器轻轻吹打充分混匀。置于温室放置5min。
(2)PCR富集
(a)将灭菌PCR管置于冰浴中，依次添加各反应组分：ddH 2O，4μl；步骤1产物，25μl；5×TAB，10μl；P5(10μM)，1μl；N7XX，5μl；TAE，1μl。
使用试剂盒为：TruePrep TM Index Kit V2 for
Figure PCTCN2021119166-appb-000002
(Vazyme#TD202)，具体实验步骤参照试剂盒使用说明。
(b)使用移液器轻轻吹打充分混匀，将PCR管置于PCR仪中进行如下反应：72℃3min，98℃预变性30sec；98℃变形15sec，60℃退火30sec；72℃延伸3min，共11个循环；4℃保存。
(3)扩增产物长度分选、纯化
使用AMPure XP磁珠进行长度分选和纯化。起始PCR产物体积应为50μl。因PCR过程中样品挥发会导致产物体积不足50μl，进行下面操作之前必须使用灭菌蒸馏水将体积补齐至50μl，否则分选长度会与预期不一致。分选过程中，两轮磁珠使用量(R1和R2)如下：
第一轮AMPure XP磁珠用量R1＝30.0μl(0.60×)第二轮AMPure XP磁珠用量R2＝7.5μl(0.15×)。
其中，“×”数均根据PCR产物体积计算而得，如“0.60×”表示0.60×50μl＝30.0μl。
(a)涡旋震荡混匀AMPure XP磁珠并吸取R1体积至50μl PCR产物中，使用移液器轻轻吹打10次充分混匀。室温孵育5分钟。
(b)将反应管短暂离心并置于磁力架中分离AMPure XP磁珠和液体。待溶液澄清(约5分钟)小心转移上清至干净EP管中，丢弃磁珠。
(c)涡旋震荡混匀AMPure XP磁珠并吸取R2体积至上清中，使用移液器轻轻吹打10次充分混匀。室温孵育5分钟。
(d)将反应管短暂离心并置于磁力架中分离AMPure XP磁珠和液体。待溶液澄清(约5分钟)小心移除上清。
(e)保持EP管始终处于磁力架中，加入200μl新鲜配制的80％乙醇漂洗AMPure XP 磁珠。室温孵育30秒后小心移除上清。
(f)重复步骤5，总计漂洗两次。
(g)保持EP管始终处于磁力架中，开盖空气干燥AMPure XP磁珠10分钟。
(h)将EP管从磁力架中取出，加入13μl灭菌超纯水洗脱。涡旋振荡或使用移液器轻轻吹打充分混匀。将反应管短暂离心并置于磁力架中分离AMPure XP磁珠和液体。待溶液澄清(约5分钟)小心吸取12μl上清至灭菌EP管中，获得测序文库，置于-20℃保存。
实施例3
不同配比裂解液的优化。
同实施例2，带有分子标签的细胞落板后，使用DPBS溶液洗去多余的分子标记磁珠，分别向三个微孔板中加入三种不同配方的裂解液，依次为对照裂解液(0.1M Tris-HCl pH 7.5，0.5M LiCl，1％SDS，10mM EDTA和5mM dithiothreitol)、裂解液20(ddH 2O，1％SDS，20％甲酰胺和3×SSC)、裂解液50(ddH 2O，1％SDS，50％甲酰胺和3×SSC)，充分裂解孵育30分钟，后续步骤同实施例2、3。其中SDS起主要的裂解作用，甲酰胺为促进核酸分子的杂交，SSC辅助提升杂交效率。基因测序文库构建好送至瑞普公司进行测序，采用HiSeq2500PE125测序策略。对测序结果进行分析发现，裂解液50显著性提升了UMI大于500的细胞数量和平均基因数(图7)，大幅提高了平台的反应效率。
实施例4
小鼠睾丸细胞的分析。
取500万个消化好的小鼠按照实施例2的步骤进行建库、获得测序文库。基因测序文库构建好送至瑞普公司进行测序，采用HiSeq2500PE125测序策略。测序返回的数据经过拆分筛选比对，得到基因表达谱。将这个矩阵文件导入R语言分析，就能将矩阵数据转换为可视化的图。图8为小鼠睾丸细胞tSNE分析结果，显示小鼠睾丸可以分为14个亚群。
实施例5
本发明同样可以应用于以微流控为主的单细胞测序平台。通过实施例2中所述方法获得带有一轮细胞标签的固定细胞后，利用10×公司铬芯片E(10×Genomics#2000121)进行反应。进口1注入75μl混合有连接体系的固定细胞(10μl细胞，50.5μl无酶水，7.5μl T4连接缓冲液，3μl T4连接酶，1.5μl 10×Reducing Agent B和2.5μl 100mM桥连引物)，进口2注入40μl含有裂解液的单细胞ATAC凝胶微珠(10×Genomics#2000132，微珠序列按照实施例1中所述)，进口3注入240μl的Partitioning Oil(10×Genomics#220088)。随后将乳液包裹的微珠置于37℃孵育1.5小时，使连接反应充分。反应结束后，收集微珠依照实施例2中所述方案进行外切、单链化、二链合 成反应，最终通过PCR扩增、纯化获得cDNA文库。
实施例6
染色质转座酶可及性超高通量单细胞测序。
将实施例1中标签磁珠与桥连引物退火。将磁珠与桥连引物(桥连引物序列：5’-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA-3’)混匀，梯度退火，形成粘性末端。没有结合桥连引物的序列使用核酸外切酶EXO I切除。外切完成后，用150μl的TE-SDS、TE-TW洗一遍，最后用TE-TW重悬，4℃保存备用。
1、特异性分子标签转座酶包埋复合物的制备
Tn5转座酶裸酶购自南京诺唯赞生物科技有限公司。转座酶及包埋缓冲液由南京诺唯赞生物科技有限公司生产的(Vazyme)Tn5 Transposome(S111)试剂盒提供。
(1)包埋核酸序列如下表2所示。
表2
Figure PCTCN2021119166-appb-000003
其中，特异标签片段包含的特异性分子标签10×N为细胞标签序列的核心序列，对应每种Tn5复合物的这个核心序列均不同，由于每个位点有A/T/C/G这4种选择，所以10×N的序列可以有4 10种选择。N表示A/T/C/G中的任一种，为随机合成。
(2)96种带特异分子标签寡核苷酸序列退火(P7 adapter片段+Mosaic Ends片段退火得到通用型序列，即图9中接头中的P7 Adapter-Mosaic Ends/Mosaic Ends；特异标签片段+Mosaic Ends片段退火得到特异性分子标签序列，即图9中的搭桥序列2-细胞标签4-Mosaic Ends/Mosaic Ends)。96孔中每孔含两种包埋片段，包括孔间相同的P7 adapter片段+Mosaic Ends片段以及孔间特异的特异标签片段+Mosaic Ends片段。
(3)将Tn5裸酶和包埋缓冲液均匀加入到96孔板中，随后每孔分别加入步骤(2)中96种标签接头冻存液，吹打5次，在30℃孵育1h后于-20℃冰箱保存(图9)。
2、新鲜组织细胞核提取
取50mg新鲜组织样本，液氮研磨成粉，迅速转移到预冷的1.5ml EP管内。用1ml裂解液(ddH 2O，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl 2，1％BSA，0.1％Tween-20，0.1％IGEPAL CA-630，0.01％洋地黄皂苷)重悬，冰上裂解3min。裂解后用RSBT缓冲液(ddH 2O，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl 2，1％BSA，0.1％Tween-20)重悬，过滤去掉组织残渣。细胞核用1％甲醛室温固定10分钟。用甘氨酸终止固定交联。配制冻存液(ddH 2O，50mM Tris-HCl pH8.0，25％甘油，5mM Mg(OAc) 2，0.1mM EDTA)，取975μl冻存液加5μl 5mM DTT和20μl 50×protease inhibitor cocktail，重悬细胞核后可-80冻存。或者用1ml RSBT缓冲液悬浮细胞核过滤 后计数备用。冻存样本复苏时将样品取出，置于放37℃烘箱解冻2min，500g/5min离心，弃上清，用200μl裂解液重悬，置冰上3min。用RSBT清洗过滤后计数备用。
3、人293T和鼠3T3混合细胞的细胞核提取
小鼠成纤维细胞(3T3)和人胚肾细胞(293T)各200万混匀后用PBS洗一遍，转移至1.5ml EP管中，随后用1ml裂解液(ddH 2O，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl 2，1％BSA，0.1％Tween-20，0.1％IGEPAL CA-630，0.01％洋地黄皂苷)重悬，冰上裂解3min。裂解后用RSBT缓冲液(ddH 2O，10mM Tris-HCl pH7.4，10mM NaCl，3mM MgCl 2，1％BSA，0.1％Tween-20)清洗两次。细胞核用1％甲醛室温固定10分钟。用甘氨酸终止固定交联。配制冻存液(ddH 2O，50mM Tris-HCl pH8.0，25％甘油，5mM Mg(OAc) 2，0.1mM EDTA)，取975μl冻存液加5μl 5mM DTT和20μl 50×protease inhibitor cocktail，重悬细胞核后可-80冻存。或者用1ml RSBT缓冲液悬浮细胞核过滤后计数备用。冻存样本复苏时将样品取出，置于放37℃烘箱解冻2min，500g/5min离心，弃上清，用200μl裂解液重悬，置冰上3min。用RSBT清洗过滤后计数备用。
4、人293T和鼠3T3混合细胞的Tn5预标记
取制备细胞核，用1ml RSBT重悬过滤并计数。配制2×TD缓冲液(ddH 2O，20mM Tris-HCl pH7.6，10mM MgCl 2，20％二甲基甲酰胺)，配制tagmentation缓冲液(每孔22.5μl，包括12.5μl 2×TD buffer，9.5μl 1×DPBS，0.25μl 1％洋地黄皂苷，0.25μl 10％Tween-20)，用tagmentation缓冲液重悬细胞核，分装至96孔板中，每孔约为1万细胞核。取实施例3中的转座酶包埋复合物加入到含细胞核的96孔板中，每孔2.5μl，置于55℃反应30min。反应结束后，每孔加入25μl 2×终止液(25ml 40mM EDTA，3.9μl 6.4M Spermidine)，37℃静置15min。收集液体混合至15ml离心管，500g/5min，弃上清，1ml RSBT清洗一次。计数，将细胞核分装至1.5ml EP管中，每管50万细胞核，20μl RSBT重悬，再加入30μl PNK反应液(5μl 10×PNK buffer，5μl 10mM ATP，10μl ddH 2O，l0μl PNK酶)，37℃孵育30min，反应完成后离心，弃上清。RSBT清洗两次后置冰上备用。
5、人293T和鼠3T3混合细胞的微孔板ATAC测序
取Tn5标记细胞核。将细胞核悬液滴加入微孔板中，利用离心机短暂离心使80-90％的细胞核落入微孔中，每个孔捕获0-10个细胞核，通过Tn5上的细胞标签4对同一孔内的细胞DNA片段进行区分。将搭桥引物和标记磁珠退火，使标记磁珠上的片段形成粘性末端。向落有细胞核的微孔板中加入20万个分子标记磁珠，置于磁铁上轻轻混匀，使磁珠覆盖99％以上的微孔，使用RSBT溶液洗去多余的分子标记磁珠。向铺满磁珠的微孔板中缓慢滴加200μL裂解液(100μl 10％SDS，40μl蛋白酶K，100μl 10×T4 buffer，200μl 50％PEG 8000(质量体积比)，560μl 10mM Tris-HCl pH8.0)，室温裂解孵育30分钟，使磁珠上的搭桥序列和细胞核上的搭桥引物充分互补杂交。孵育完成后，将微 孔板倒置于磁铁上，收集带有分子标记-DNA复合物的磁珠，转移至1.5ml EP管中，6×SSC清洗两次，随后用50mM Tris pH8.0再洗一遍，向装有分子标记磁珠的EP管中加入连接混合液50μL(2μl T4连接酶，5μL T4缓冲液，2μL dNTP，10μL 50％PEG8000(质量体积比)，31μL ddH 2O)，置于25℃反应1.5小时。连接反应完成后，把分子标记磁珠在磁力架上用TE-SDS、TE-TW、10mM Tris pH8.0各洗一遍，用100μL延伸体系(20μL5×RT buffer，10μL dNTP，2.5μL Klenow聚合酶，20μl 50％PEG(质量体积比)和47.5μl ddH 2O)悬浮磁珠，置于37℃反应1小时。反应结束后，把分子标记磁珠在磁力架上用TE-SDS、TE-TW、10mM Tris pH8.0各洗一遍，用500μL 0.1M的NaOH悬浮磁珠，室温孵育5分钟进行单链化。然后用TE-TW洗两次、10mM Tris pH8.0洗一遍，用index P5和index P7引物将目的片段从磁珠上富集出来，并用诺唯赞纯化磁珠纯化PCR产物，获得300-500bp的产物。
上述反应具体流程见图10，文库大小分布在300-500bp左右，见图11。
将上述方法构建基因测序文库，基因测序文库构建好后使用MGI/Illumina二代测序仪进行测序。测序返回的数据经过拆分筛选比对，得到cell by peak矩阵。将这个矩阵文件导入R语言分析，就能将矩阵数据转换为可视化的图。由图12-17可知，存在极少量双细胞污染(图12)，片段大小符合预期(图13)，peak分布正常(图14)，TSS富集(图15)，小鼠3T3细胞群体和单细胞ATAC peak分布(图16和图17)，测序质量可以达到单细胞ATAC高通量测序水平。
建库使用引物序列如下表3所示。其中N表示A/T/C/G中的任一种，为随机合成。S表示末端碱基的硫代修饰，提高末端碱基的稳定性。
表3
Figure PCTCN2021119166-appb-000004
实施例7
本发明染色质转座酶可及性超高通量单细胞测序同样可以应用于以微流控为主的单细胞测序平台。通过实施例6中所述方法获得带有一轮细胞标签的固定细胞核后，利用10×公司铬芯片E(10×Genomics#2000121)进行反应。进口1注入75μl混合有连接体系的固定细胞(10μl细胞，50.5μl无酶水，7.5μl T4连接缓冲液，3μl T4连接酶，1.5μl 10×Reducing Agent B和2.5μl 100mM桥连引物)，进口2注入40μl含有裂解液的单细胞ATAC凝胶微珠(10×Genomics#2000132，微珠序列按照实施例1中所述)，进口3注入240μl的Partitioning Oil(10×Genomics#220088)。随后将乳液包裹的微珠置于37℃孵育1.5小时，使连接反应充分。反应结束后，收集微珠依照实施例7中所述方案进行外切、单链化、二链合成反应，最终通过PCR扩增、纯化获得基因测序文库，测序和生信分析。基因测序文库按照实施例7中的流程进行测序和生信分析。
实施例8
本发明染色质转座酶可及性超高通量单细胞测序同样可以应用于双端都标记的Tn5包埋方法。
按照实施例6中方法进行包埋，但包埋时，96孔中每孔含1种包埋片段，为孔间特异的特异标签片段+Mosaic Ends片段，特异标签片段+Mosaic Ends片段退火得到特异性分子标签序列，即图9中的搭桥序列2-细胞标签4-Mosaic Ends/Mosaic Ends。
然后，按照实施例6中方案对细胞核进行标记，标记后的细胞核进行微孔板捕获，连接，延伸。加入500μL 0.1M的NaOH溶液处理5分钟进行单链化，再加入P7adapter-Mosaic Ends引物(即包括了P7 adapter和Mosaic Ends片段序列的引物)进行延伸。延伸完成后通过高温变性收集上清单链产物，去掉磁珠。用index P5和index P7引物PCR扩增、纯化获得基因测序文库(如图18)。基因测序文库按照实施例7中的流程进行测序和生信分析。

Claims (10)

  1. 一种超高通量单细胞测序方法，其特征在于，包括以下步骤：
    (1)准备如下试剂：
    a)分子标记微珠，所述分子标记微珠包括微珠本体和耦联的分子标记序列，该分子标记序列包括顺序排列的：
    通用引物序列，作为PCR扩增时的引物结合区域；
    第一细胞标签序列；
    第一搭桥序列；
    b)反转录序列，用于细胞内反转录，所述反转录序列包括顺序排列的：
    第二搭桥序列；
    第二细胞标签序列，与第一细胞标签序列配合组成细胞标签序列，所述细胞标签序列用于标识所构建测序文库中各序列所对应的mRNA取自的细胞；
    分子标签序列，用于标识所构建测序文库中各序列所对应的mRNA；
    多聚T尾，用于与细胞中带有poly-A序列的mRNA互补配对；
    c)桥连引物，用于将上述a)中的标记序列与b)中的反转录序列进行连接，所述桥连引物两端具有分别与所述第一搭桥序列和第二搭桥序列互补配对的序列；
    (2)取待测序的细胞样品，加入反转录序列进行细胞内反转录，使反转录序列的多聚T尾端连上将细胞内mRNA序列反转录后得到的cDNA序列，获得反转录序列-cDNA序列；
    (3)将步骤(2)经细胞内反转录后的细胞通过微孔板技术或微流控技术使一个分子标记微珠与一个或多个细胞处于分隔的空间中，并在裂解液作用下裂解细胞，孵育，通过桥连引物分别与所述第一搭桥序列和第二搭桥序列互补配对，然后使用连接酶连接，使第一搭桥序列和第二搭桥序列连接获得与微珠耦联的分子标记序列-反转录序列-cDNA序列；
    (4)收集耦联有分子标记序列-反转录序列-cDNA序列的微珠，进行PCR扩增获得带有第一细胞标签序列、第二细胞标签序列和分子标签序列的cDNA序列；
    (5)将步骤(4)获得的产物构建cDNA测序文库，然后进行高通量测序，获得上百万个单细胞的特异性转录组信息。
  2. 一种超高通量单细胞测序方法，其特征在于，包括以下步骤：
    (1)准备如下试剂：
    a)分子标记微珠，所述分子标记微珠包括微珠本体和耦联的分子标记序列，该分子标记序列包括顺序排列的：
    通用引物序列，作为PCR扩增时的引物结合区域；
    第一细胞标签序列；
    第一搭桥序列；
    b)特异性分子标签转座酶包埋复合物，包括Tn5转座酶和特异性分子标签序列，其中特异性分子标签序列包括顺序排列的：
    第二搭桥序列；
    第二细胞标签序列，与第一细胞标签序列配合组成细胞标签序列，所述细胞标签序列用于标识所构建测序文库中各序列来源的细胞；
    Mosaic Ends序列，用于与Tn5转座酶结合，所述Mosaic Ends序列为双链结构，其中一条链与第二细胞标签序列连接；
    c)桥连引物，用于将上述a)中的分子标记序列与b)中的特异性分子标签序列进行连接，所述桥连引物两端具有分别与所述第一搭桥序列和第二搭桥序列互补配对的序列；
    (2)取待测序的细胞样品，提取细胞核；
    (3)向步骤(2)提取的细胞核中加入特异性分子标签转座酶包埋复合物进行转座反应；
    (4)将经过转座反应后的细胞核通过微孔板技术或微流控技术使一个分子标记微珠与一个或多个细胞核处于分隔的空间中，并在裂解液作用下裂解细胞核，孵育，通过桥连引物分别与所述第一搭桥序列和第二搭桥序列互补配对，然后使用连接酶连接，使第一搭桥序列和第二搭桥序列连接获得与微珠耦联的分子标记序列-特异性分子标签序列-染色质转座酶可及性基因组序列；
    (5)收集耦联有分子标记序列-特异性分子标签序列-染色质转座酶可及性基因组序列的微珠，进行PCR扩增获得带有第一细胞标签序列、第二细胞标签序列和特异性分子标签序列的染色质转座酶可及性基因组序列；
    (6)将步骤(5)获得的产物构建染色质可及性测序文库，然后进行高通量测序，获得上百万个单细胞的特异性基因组开放性信息。
  3. 根据权利要求1或2所述超高通量单细胞测序方法，其特征在于，所述微珠与分子标记序列耦联方式为：分子标记序列5’端的核苷酸的C6位上使用胺基取代羟基，微珠表面修饰有羧基，通过羧基与氨基缩合耦联。
  4. 根据权利要求1或2所述超高通量单细胞测序方法，其特征在于，所述第一细胞标签序列包括多个特异性片段，所述第二细胞标签序列包括至少一个特异性片段，不同位置的特异性片段选自相同的或不同的特异性片段库，所述第一细胞标签序列和第二细胞标签序列利用所述特异性片段的排列组合方式不同标识细胞。
  5. 根据权利要求1或2所述超高通量单细胞测序方法，其特征在于，所述分子标记微珠的制备方法包括以下步骤：
    (1)用于合成分子标记序列的引物根据特异性片段的数量分为多条引物，每条引物包含一条特异性片段，各引物之间具有用于搭桥连接、互补的接头序列，其中对应于分子标记序列5’端的那条引物还包括所述通用引物序列，对应于分子标记序列3’端的那条引物还包括所述第一搭桥序列；
    (2)将对应于分子标记序列5’端的那条引物与微珠本体耦联，然后通过PCR方法将剩余引物依次退火、延伸，从5’端到3’依次串接分子标记序列的其余特异性片段，制备得到所述分子标记微珠。
  6. 根据权利要求5所述超高通量单细胞测序方法，其特征在于，所述分子标记序列为：5’-TTTAGGGATAACAGGGTAATAAGCAGTGGTATCAACGCAGAGTACGTNNNNNNCGACTCACTACAGGGNNNNNNTCGGTGACACGATCGNNNNNNTCGTCGGCAGCGTC-3’，其中，N表示A/T/C/G中的任一种，为随机合成。
  7. 根据权利要求1或2所述超高通量单细胞测序方法，其特征在于，待测序的细胞样品中含有2种或2种以上的细胞。
  8. 根据权利要求1或2所述超高通量单细胞测序方法，其特征在于，所述微珠本体为磁珠，
    将经细胞内反转录后的细胞或者经转座反应后的细胞核加入到微孔板中，然后加入分子标记微珠，微孔板中的微孔直径大小为刚好容纳一个分子标记微珠和一个或多个细胞/细胞核；
    细胞或细胞核加入微孔板中的落孔率控制在大于80％；分子标记微珠加入微孔板中的落孔率大于99％。
  9. 如权利要求8所述的超高通量单细胞测序方法，其特征在于，所述微孔板中微孔深度30-160μm、微孔直径20-150μm；所述微珠本体的直径为20-145μm。
  10. 如权利要求8所述的超高通量单细胞测序方法，其特征在于，所述微孔板的制备方法为：
    (1)在硅片上刻蚀出微孔作为初始模具；
    (2)在初始模具上浇注聚二甲基硅氧烷，成型后取下聚二甲基硅氧烷成为具有微柱的第二次模具；
    (3)在第二次模具上浇注热熔的质量体积比为4％～6％的琼脂糖，冷却成型后，取下琼脂糖即为所述微孔板。
PCT/CN2021/119166 2021-05-12 2021-09-17 一种超高通量单细胞测序方法 WO2022237035A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/785,045 US20240182962A1 (en) 2021-05-12 2021-09-17 Ultra-high-throughput single cell sequencing method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110517750.6A CN113106150B (zh) 2021-05-12 2021-05-12 一种超高通量单细胞测序方法
CN202110517750.6 2021-05-12
CN202110911428.1 2021-08-09
CN202110911428.1A CN113604545B (zh) 2021-08-09 2021-08-09 一种超高通量单细胞染色质转座酶可及性测序方法

Publications (1)

Publication Number Publication Date
WO2022237035A1 true WO2022237035A1 (zh) 2022-11-17

Family

ID=84028797

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/119166 WO2022237035A1 (zh) 2021-05-12 2021-09-17 一种超高通量单细胞测序方法

Country Status (2)

Country Link
US (1) US20240182962A1 (zh)
WO (1) WO2022237035A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116564415A (zh) * 2023-07-10 2023-08-08 深圳华大基因科技服务有限公司 流式测序分析方法、装置、存储介质及计算机设备
CN118272508A (zh) * 2023-06-28 2024-07-02 浙江大学 基于单端接头转座酶的单细胞基因组测序试剂盒及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106498040A (zh) * 2016-10-12 2017-03-15 浙江大学 一种分子标记微珠及基于该分子标记微珠的高通量单细胞测序方法
CN110684829A (zh) * 2018-07-05 2020-01-14 深圳华大智造科技有限公司 一种高通量的单细胞转录组测序方法和试剂盒
CN111088250A (zh) * 2019-12-25 2020-05-01 中国科学院苏州生物医学工程技术研究所 一种mRNA捕获序列、捕获载体的合成方法、高通量单细胞测序文库制备方法
CN113106150A (zh) * 2021-05-12 2021-07-13 浙江大学 一种超高通量单细胞测序方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106498040A (zh) * 2016-10-12 2017-03-15 浙江大学 一种分子标记微珠及基于该分子标记微珠的高通量单细胞测序方法
CN110684829A (zh) * 2018-07-05 2020-01-14 深圳华大智造科技有限公司 一种高通量的单细胞转录组测序方法和试剂盒
CN111088250A (zh) * 2019-12-25 2020-05-01 中国科学院苏州生物医学工程技术研究所 一种mRNA捕获序列、捕获载体的合成方法、高通量单细胞测序文库制备方法
CN113106150A (zh) * 2021-05-12 2021-07-13 浙江大学 一种超高通量单细胞测序方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JUNYUE CAO, JONATHAN S. PACKER, VIJAY RAMANI, DARREN A. CUSANOVICH, CHAU HUYNH, RIZA DAZA, XIAOJIE QIU, CHOLI LEE, SCOTT N. FURLAN: "Comprehensive single-cell transcriptional profiling of a multicellular organism", SCIENCE, vol. 357, no. 6352, 18 August 2017 (2017-08-18), US , pages 661 - 667, XP055624798, ISSN: 0036-8075, DOI: 10.1126/science.aam8940 *
ROSENBERG ALEXANDER B., ROCO CHARLES M., MUSCAT RICHARD A., KUCHINA ANNA, SAMPLE PAUL, YAO ZIZHEN, GRAYBUCK LUCAS T., PEELER DAVID: "Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding - suppl. material", SCIENCE, vol. 360, no. 6385, 13 April 2018 (2018-04-13), US , pages 176 - 182, XP055803532, ISSN: 0036-8075, DOI: 10.1126/science.aam8999 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118272508A (zh) * 2023-06-28 2024-07-02 浙江大学 基于单端接头转座酶的单细胞基因组测序试剂盒及其应用
CN116564415A (zh) * 2023-07-10 2023-08-08 深圳华大基因科技服务有限公司 流式测序分析方法、装置、存储介质及计算机设备
CN116564415B (zh) * 2023-07-10 2023-10-17 深圳华大基因科技服务有限公司 流式测序分析方法、装置、存储介质及计算机设备

Also Published As

Publication number Publication date
US20240182962A1 (en) 2024-06-06

Similar Documents

Publication Publication Date Title
US11555216B2 (en) Methods and kits for labeling cellular molecules
CN114958996B (zh) 一种超高通量单细胞测序试剂组合
JP6324962B2 (ja) 標的ｒｎａ枯渇化組成物を調製するための方法およびキット
US20230340567A1 (en) In situ combinatorial labeling of cellular molecules
CN113604545B (zh) 一种超高通量单细胞染色质转座酶可及性测序方法
WO2022237035A1 (zh) 一种超高通量单细胞测序方法
CN105658813B (zh) 包括选择和富集步骤的染色体构象捕获方法
CN110886021B (zh) 一种单细胞dna文库的构建方法
CN115176026A (zh) Rna寡核苷酸的测序方法
CN114729349A (zh) 条码化核酸用于检测和测序的方法
CN114574569B (zh) 一种基于末端转移酶的基因组测序试剂盒和测序方法
CN117089597A (zh) 一种单细胞文库构建测序方法及其应用
CN111801428B (zh) 一种获得单细胞mRNA序列的方法
CN115537408A (zh) 一种单细胞多组学文库及其构建方法
WO2023237180A1 (en) Optimised set of oligonucleotides for bulk rna barcoding and sequencing
CN117651611A (zh) 生物分子的高通量分析

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 17785045

Country of ref document: US

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21941602

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21941602

Country of ref document: EP

Kind code of ref document: A1