WO2023155135A1 - 单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒 - Google Patents

单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒 Download PDF

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WO2023155135A1
WO2023155135A1 PCT/CN2022/076852 CN2022076852W WO2023155135A1 WO 2023155135 A1 WO2023155135 A1 WO 2023155135A1 CN 2022076852 W CN2022076852 W CN 2022076852W WO 2023155135 A1 WO2023155135 A1 WO 2023155135A1
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cdna
library
round
cell
cells
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French (fr)
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王明月
刘龙奇
刘传宇
郁葱
黄亚灵
林秀妹
袁月
王智锋
魏小雨
王晓山
向蓉
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深圳华大生命科学研究院
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    • 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
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    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention relates to the technical field of molecular biology, in particular to a method for constructing a single-cell transcriptome sequencing library, a sequencing method, and a kit for preparing a single-cell transcriptome library.
  • a single cell is the fundamental unit of structure and function in biological systems.
  • conventional methods based on large numbers of cells for analyzing the molecular content of biological samples mask the heterogeneity of cells in biological systems.
  • the first single-cell sequencing technology appeared in 2009. Since then, more and more studies have been conducted at the single-cell level. Analyzing the transcriptome of a single cell has become an effective strategy to solve biological heterogeneity.
  • single-cell transcriptome sequencing can be used to classify cell types and detect cell states.
  • methods for single-cell RNA sequencing sc-RNA-seq
  • conventional droplet microfluidics methods to obtain single-cell RNA-sequencing data are expensive, with costs growing linearly with the number of cells processed.
  • the density of the cells needs to be adjusted to a very low concentration to load the droplets. This leads to extremely low efficiency of capturing cells in a single experiment, and virtually increases the cost of single-cell RNA sequencing.
  • RNA labeling cells ahead of time is a great strategy for differentiating cells within the same droplet. And there are already some methods that can realize the pre-labeling of RNA in cells.
  • the most commonly used method is to carry out reverse transcription of RNA in the nucleus, using a labeled oligo-thymine (oligo-dT) primer to first hybridize with the polyadenylic acid (poly A) tail of the mRNA, and then in the Under the action of reverse transcriptase, RNA is used as template to reverse transcribe into cDNA.
  • the cDNA product obtained in this way carries the label on the oligo-dT primer, and by this method, the intracellular transcript can be marked in advance.
  • the method of labeling in advance needs to realize the reverse transcription at the cell or nucleus level.
  • the article published by Cao et al. (Comprehensive single-cell transcriptional profiling of a m ⁇ Lticell ⁇ Lar organism; Science; 18Aug 2017: Vol.357, Issue 6352, pp.661-667) disclosed sci-RNA-seq technology and sci-RNA-seq3 technology. In both techniques, the cells are first divided equally into 96 or 384 well plates, and a labeled oligo-dT primer is added to each well, that is to say, there are 96 or 384 labeled in the first round. mark.
  • droplet microfluidics-based single-cell sequencing Another method for early labeling is droplet microfluidics-based single-cell sequencing (drop-seq).
  • the article published by Macosko et al. discloses a method using A Single-Cell Sequencing Method for ⁇ L Droplets.
  • water-in-oil droplets are used to wrap cells and micro-particles based on droplet microfluidic technology, and a droplet containing both a cell and a micro-particle is obtained.
  • the droplets also contain lysate, and the beads contain a large number of DNA sequences with specific molecular markers called barcodes.
  • the cells in the droplets are affected by the lysate, releasing a large amount of mRNA, and at the same time, the mRNA can be captured by the microbeads.
  • the droplets are broken.
  • the mRNA is reverse transcribed into cDNA and further amplified.
  • the cDNA is broken by treating it with a breaking enzyme, and sequencing adapters are added to both ends, and the adapter-added cDNA is subsequently sequenced.
  • the density of the bead phase and the cell phase must be reduced to a very low concentration. Therefore, it is difficult for this method to achieve ultra-high-throughput single-cell sequencing of more than 10,000 cells per experiment, which virtually increases the cost of each cell.
  • the main purpose of the present invention is to provide a method for constructing a single-cell transcriptome sequencing library, a sequencing method, and a kit for preparing a single-cell transcriptome library to solve the problem of low construction efficiency of a single-cell transcriptome sequencing library in the prior art.
  • the present invention proposes a method for constructing a single-cell transcriptome sequencing library.
  • the method includes the following steps:
  • the oligo-dT primer containing the tag is used for in situ reverse transcription to generate a full-length first-strand cDNA with the first label;
  • the 3' sequence includes in turn: a. a magnetic bead capture region, which is a complementary region to the oligonucleotide in the second label on the magnetic bead; b. a single-molecule label for identifying a single cDNA transcript; c . the first round of labeling for distinguishing different cells or nuclei; and d.
  • a poly T sequence for capturing mRNA; thereby making the 5'-end of the obtained full-length first-strand cDNA with the first label follow In order from 5' to 3' include: magnetic bead capture region, which is a complementary region to the oligonucleotide in the second label on the magnetic bead; single molecule label; first round label and poly T sequence , wherein the magnetic beads are capable of recognizing the magnetic bead capture region in the first label.
  • a reverse transcriptase with terminal transferase activity is used to perform in situ reverse transcription, and the in situ reverse transcription system also includes a template displacement primer (Tn-TSO), preferably with terminal transfer
  • Tn-TSO template displacement primer
  • the enzymatically active reverse transcriptase is selected from Maxima H enzyme, SSII enzyme or SSIV enzyme; preferably, the template replacement primer is designed to include guanine at the 3'-end, preferably, the number of guanine is 3.
  • the in situ reverse transcription system also contains glycerol, preferably, the final concentration of glycerol in the in situ reverse transcription system is 5% to 10%; preferably, the construction method also includes: Carry out fixation and/or permeabilization pretreatment to the cells or nuclei in the single-cell suspension; More preferably, use an organic solvent to perform fixation and/or permeabilization pretreatment; More preferably, the organic solvent is methanol or paraformaldehyde; More preferably Further preferably, the working concentration of methanol is 50%-100%, and the working concentration of paraformaldehyde is 1%-4%.
  • step 2) by controlling the density of cells or nuclei in the single-cell suspension to be 100-1000 per ⁇ L, and the density of magnetic beads to be 2000-5000 per ⁇ L; preferably, the density of cells or nuclei The density was 500 per ⁇ L, and the density of the magnetic beads was 3000 per ⁇ L.
  • the PCR reaction system for the synthesis of the second strand cDNA in step 2) includes: primers and PCR reagents that are complementary to the 3'-end of the first strand cDNA and magnetic bead primers; preferably, simultaneously with the full-length
  • the primer complementary to the 3'-end of the first-strand cDNA and the magnetic bead primer is a Tn primer.
  • the magnetic beads in step 2) are magnetic beads with a second label.
  • the second label includes in order of distance from the magnetic beads: Tn primers, a second round of labels, and A cDNA capture sequence that recognizes and captures the full-length first-strand cDNA; more preferably, the second round of tags includes a second round of first tags and a second round of second tags; further preferably, a second round of first tags and second rounds of tags A first linker is also included between the round of second labels; more preferably, a second linker is also included between the Tn primer and the magnetic beads.
  • step 5) includes: performing fragmentation library construction on the secondary amplified cDNA obtained in step 4) to obtain a chained cDNA library, which is a single-cell transcriptome sequencing library; preferably , further preparing the chained cDNA library into a circular cDNA library, and the circular cDNA library is a single-cell transcriptome sequencing library; preferably, preparing the chained cDNA library into a circular cDNA library includes: converting the chained cDNA library into a circular cDNA library The double-stranded cDNA is melted under the action of the circularization auxiliary sequence to obtain the melted product; the melted product is ligated by DNA ligase to obtain the single-stranded circularized product; Circularized single-stranded cDNA and double-stranded cDNA to obtain a circularized cDNA library; more preferably, after enzymatic digestion and degradation of the single-stranded circularized product, the degraded single-stranded circularized product is
  • the present invention proposes a sequencing method for a single-cell transcriptome library, characterized in that the sequencing method includes:
  • the present invention proposes a kit for preparing a single-cell transcriptome library, characterized in that the kit includes at least one of the following items: a tag-containing oligomeric -dT primer, template switching primer, reverse transcriptase, glycerol, fixation and/or permeabilization reagents, primers complementary to both the 3'-end of the full-length first-strand cDNA and magnetic bead primer, magnetic Beads, strand cDNA library construction reagents and circular cDNA library construction reagents; preferably, the oligo-dT primers containing the label include in order from 5' to 3': a.
  • magnetic bead capture region which is compatible with magnetic Complementary regions complementary to oligonucleotides in a second label on the bead; b. single-molecule labeling to identify individual cDNA transcripts; c. first-round labeling to differentiate between different cells or nuclei; and d.
  • the reverse transcriptase is a reverse transcriptase with terminal transferase activity, more preferably the reverse transcriptase with terminal transferase activity is selected from Maxima H enzyme, SSII enzyme or SSIV enzyme; preferably
  • the template switching primer is designed to contain guanine, preferably, the number of guanine is 3; preferably, the final concentration of glycerol in the in situ reverse transcription system is 5% to 10%; preferably, the fixed and / or the permeabilization solvent is an organic solvent, more preferably, the organic solvent is methanol or paraformaldehyde, more preferably, the working concentration of methanol is 50% to 100%, and the working concentration of paraformaldehyde is 1% to 4%.
  • the primer complementary to the 3'-end of the full-length first strand cDNA and the magnetic bead primer is a Tn primer; preferably, in the magnetic beads with the second label, the second label is according to the distance from the magnetic bead
  • the sequence from near to far includes: Tn primers, the second round of tags and the cDNA capture sequence used to capture the first strand cDNA, more preferably, the second round of tags includes the second round of first tags and the second round of second Label, further preferably, a first linker is also included between the first label of the second round and the second label of the second round, even more preferably, a second linker is also included between the Tn primer and the magnetic bead; preferably, a chain
  • the cDNA library construction reagent is used to construct a fragmented cDNA library after reverse transcription and amplification to obtain a chain cDNA library; preferably, the circular cDNA library construction reagent is used to prepare the chain cDNA library into a circular cDNA
  • the application of the technical scheme of the present invention realizes the overloading of cells or nuclei after in situ reverse transcription and pretreated magnetic beads, and provides an efficient, flexible and easy-to-use large-scale scRNA sequencing method.
  • the method of the present invention is simple to operate and high in efficiency, and can efficiently construct a single-cell transcriptome sequencing library, thereby greatly improving the throughput of the single-cell transcriptome sequencing method based on droplet microfluidics, and achieving the level of 100,000 cells obtained in a single experiment .
  • the present invention saves experimental costs, thus facilitating the development of large-scale single-cell research projects.
  • Figure 1 shows a flowchart of the prior art sci-RNA-seq technology.
  • the cells are first divided into 96-well plates, and a labeled oligo-dT primer is added to each well, that is, there are 96 labels in the first round of labeling.
  • the recovered products were evenly distributed to the second 96-well plate, and similarly, a different labeled primer was added to each well.
  • the second round of marking also has a total of 96 or 384 markings.
  • a second round of labeling is done by PCR reactions.
  • Fig. 2 shows a flow chart of the single-cell sequencing technology based on droplet microfluidics in the prior art.
  • the cells and microbeads are first wrapped with water-in-oil droplets to obtain a droplet containing both a cell and a microbead.
  • Other components in the droplet include lysate, and the microbeads contain a large amount of DNA sequences with specific molecular markers called tags.
  • the cells in the droplets are split by the lysate, releasing a large amount of mRNA, and at the same time, the mRNA can be captured by the microbeads.
  • the droplets are broken.
  • the mRNA is reverse transcribed into cDNA, which is further amplified.
  • the cDNA was interrupted with a fragmentation enzyme and sequencing adapters were added to both ends. Further, the adapter-added cDNA was sequenced.
  • Fig. 3 shows a flow chart of the construction method of the single-cell transcriptome sequencing library and the sequencing method of the single-cell transcriptome library according to the present invention.
  • FIG. 4 illustrates droplet generation operations for implementing overloading, according to an embodiment of the invention. Tear off the protective film on the surface of the chip and place it in the chip groove area of the droplet generating device; insert the end of the connecting tube A on the collection cover (it is the connecting tube that contacts the bottom of the collecting tube) into the outlet hole of the chip; insert a 50mL syringe (BD , 300136) placed on the fixed frame, and adjust the push rod to the initial position. Use a flat needle to connect the syringe to the connection tube B end on the collection tube cap (which is the connection tube that does not touch the bottom of the collection tube).
  • BD 50mL syringe
  • FIG. 5 shows the results of ultra-high-throughput single-cell transcriptome sequencing using the human 293T cell line according to an embodiment of the present invention.
  • the sample is methanol-fixed human 293T cell line, and the first round of labeling in this experiment uses 24 kinds of labels.
  • nearly 20,000 usable cells were obtained from a single chip.
  • FIG. 6 shows the results of ultra-high-throughput single-cell transcriptome sequencing using human 293T and murine 3T3 mixed cell lines according to an embodiment of the present invention.
  • the sample is methanol-fixed human 293T and mouse 3T3 mixed cell line, and the first round of labeling in this experiment uses 48 kinds of labels.
  • about 30,000 usable cells were obtained from a single chip.
  • the double-pack rate of human and mouse cells was 1.59%.
  • FIG. 7 shows the results of ultra-high-throughput single-cell transcriptome sequencing using human 293T and murine 3T3 mixed cell lines according to an embodiment of the present invention.
  • the sample is methanol-fixed human 293T and mouse 3T3 mixed cell line, and the first round of labeling in this experiment uses 96 kinds of labels.
  • 80,000 usable cells were obtained from a single chip.
  • the double-pack rate of human and mouse cells is 10.65%.
  • Figure 1 shows a flowchart of the prior art sci-RNA-seq technology.
  • the traditional multi-round cell labeling method although tens of thousands of cells can be obtained in a single experiment, the throughput of a single experiment is greatly improved, thereby saving costs, however, the disadvantages of this experimental method The reason is that it is a manual operation based on orifice plates, which cannot realize automatic cell capture, which greatly increases the workload; in addition, the experimental steps are very cumbersome; and each round of cell recovery will have a certain loss, resulting in a low cell recovery rate.
  • Fig. 2 shows a flow chart of the single-cell sequencing technology based on droplet microfluidics in the prior art.
  • this traditional single-cell sequencing technology based on droplet microfluidics in order to minimize the occurrence of double particles or double packets, the density of the particle phase and the cell phase must be reduced to a very low concentration. Therefore, it is difficult for this method to achieve ultra-high-throughput single-cell sequencing of more than 10,000 cells per experiment, which virtually increases the cost of each cell.
  • Fig. 3 shows a flow chart of the construction method of the single-cell transcriptome sequencing library and the sequencing method of the single-cell transcriptome library according to the present invention.
  • the present invention provides a single-cell transcriptome sequencing library construction method and a single-cell transcriptome sequencing library based on this single-cell transcriptome sequencing library construction method. Library sequencing methods.
  • the method for constructing a single-cell transcriptome sequencing library first performs in situ reverse transcription in cells or nuclei on a single-cell suspension in a well plate to generate a full-length first-strand cDNA with a first marker, Cells or nuclei are thus marked in advance by in situ reverse transcription.
  • the number of markers for all cells in this step ranged from 24 to 384.
  • the in situ reverse-transcribed cells or nuclei and the PCR reaction system and magnetic beads for second-strand cDNA synthesis are superloaded into the droplet.
  • the droplet has 1-5 cells or nuclei, and accordingly, it is guaranteed that there are 1-10 magnetic beads in the droplet.
  • the method for sequencing a single-cell transcriptome library first constructs a single-cell transcriptome sequencing library, and performs sequencing on the single-cell transcriptome sequencing library. After obtaining high-throughput sequencing data, magnetic beads from the same droplet are first combined according to the correlation of captured fragments between magnetic beads. Cells are lysed in a droplet, cDNA is released, and magnetic beads in the droplet randomly capture the released cDNA. Therefore, the fragments captured by the magnetic beads in a droplet are all the cDNA fragments of the cells in the droplet. At the same time, the cDNA template is amplified by PCR in the droplet.
  • the copy number of the template increases, and the probability of each copied template being captured by different magnetic beads in the same droplet is equal. Therefore, even if there are several different magnetic beads in the same droplet, the fragments captured by them should be very consistent, so the consistent magnetic beads can be combined by calculating the correlation of the captured fragments of the magnetic beads. Therefore, the method according to the present invention realizes the overloading of the magnetic beads in the liquid droplets, so as to improve the utilization efficiency of the liquid droplets. Overloaded cells or nuclei in the same droplet were then distinguished based on the first round of labeling, thereby distinguishing double-packed cases at increasing cell densities.
  • a double packet refers to two or more cells in a droplet; if the double packet cannot be distinguished, we can only obtain the sum of the transcripts of these two cells from the sequencing data, and cannot obtain the transcripts of a single cell. feature. Can cause problems for single-cell downstream analysis. Therefore, it is necessary to distinguish the two-pack situation.
  • the magnetic beads are distinguished according to the pre-labeled cell label (cell barcode).
  • cell barcode the pre-labeled cell label
  • sub-cells sub-cells
  • Molecular marker (UMI) correlations combine them. Since PCR is performed on the single-molecule marker in the droplet, there will be multiple copies of the single-molecule marker in the same cell, and then multiple copies are captured by different magnetic beads in the droplet. If there are more identical single-molecule markers among several daughter cells, it means that they have captured the same cell.
  • beads within a droplet are distinguished based on single-molecule marker correlations, and consistent beads are combined.
  • sub-cells*UMI unimolecular markers
  • Jaccard index the Jacquard between sub-cells Coefficient
  • the overloading of magnetic beads in the droplet is achieved by increasing the density of the magnetic bead phase.
  • the density of the magnetic beads used in this experiment is 3000/ ⁇ L.
  • the probability of wrapping one magnetic bead in the droplet is 36.7%, and the probability of wrapping more than two magnetic beads is 30%.
  • the probability density function of the Poisson distribution used here is:
  • ⁇ is the average number of cells in each droplet, and t is a constant.
  • the oligo-dT used in the first round has a total of 96 to 384 markers, which is equivalent to labeling the cells with 96 to 384 markers in the first round.
  • the cell density is artificially controlled according to the calculation formula of Poisson distribution so that no more than 10 cells are contained in each droplet. It is equivalent to randomly selecting 10 cells from 96 to 384 types of cells, then these 10 cells have a high probability of carrying 10 different markers in the first round.
  • the magnetic beads in each droplet are labeled differently for the second round of labeling. After sequencing, cells from the same droplet can be differentiated based on two rounds of labeling.
  • the construction method of single-cell transcriptome sequencing library comprises the following steps:
  • step 2) The in situ reverse-transcribed cells or nuclei obtained in step 1) and the PCR reaction system for second-strand cDNA synthesis and magnetic beads capable of recognizing the first label are overloaded into the droplet, and the droplet PCR reaction;
  • a tag-containing oligo-dT (Indexed oligo-dT) primer is used to perform in situ reverse transcription to generate a full-length first-strand cDNA with a first marker.
  • the tagged oligo-dT primers include in order from 5' to 3':
  • the magnetic bead capture region which is a complementary region (also known as aptamer, Adaptor) complementary to the oligonucleotide in the second label on the magnetic bead, is used for the magnetic bead to capture cDNA;
  • UMI Unique molecular index
  • the first round of labeling (Round 1 Barcode) used to distinguish different cells or nuclei, which marks the cells with a round of labels to distinguish cells;
  • Poly T sequence (dT30VN) for capturing mRNA.
  • the 5'-end of the obtained cDNA product includes in order from 5' to 3': the magnetic bead capture region, which is in the second label on the magnetic bead
  • the oligonucleotide complementary complementary regions also known as aptamers, Adaptor
  • single-molecule markers also known as aptamers, Adaptor
  • first-round tags and poly-T sequences (dT30V).
  • the magnetic beads are capable of recognizing the magnetic bead capture region in the first label.
  • the in situ reverse transcription is performed using a reverse transcriptase having terminal transferase activity.
  • the reverse transcriptase having terminal transferase activity is selected from Maxima H enzyme, SSII enzyme or SSIV enzyme.
  • Cytosine (C) is added to the end of the first strand of cDNA through the action of reverse transcriptase, preferably the number of cytosine is 3.
  • the first strand of cDNA is obtained, and its structure can be expressed as: magnetic bead capture region-single-molecule label-first round label-poly T sequence-cDNA-CCC.
  • the in situ reverse transcription system further includes template-displacing primers, also known as template-displacing oligonucleotides (Template switch oligo, Tn-TSO).
  • template-displacing primers also known as template-displacing oligonucleotides (Template switch oligo, Tn-TSO).
  • the sequence of the template replacement primer is: 5'-CGTAGCCATGTCGTTCTG rGrG+G -3', wherein, "+G” represents a locked nucleotide-modified guanine, and "rG” is a guanine nucleotide,
  • the template replacement primer is designed to include guanine nucleotides (G) at the 3'-end, preferably three guanine nucleotides (Tn-TSO- rGrG+G ).
  • the template displacement primer is complementary to the cytosine on the cDNA through its guanine, that is, the CCC at the 3'-end of the obtained cDNA product (cDNA first strand) and the 3'- of Tn-TSO-rGrG+G
  • the sequence of the terminal rGrG+G is complementary, so that the template displacement primer continues to serve as a template for reverse transcription to guide the synthesis of cDNA.
  • the in situ reverse transcription system further includes glycerol.
  • the final concentration of glycerol in the in situ reverse transcription system is 5%-10%.
  • Glycerol protects the integrity of cells or nuclei.
  • glycerol can reduce the adhesion between cells and indirectly improve the efficiency of reverse transcription.
  • cells or nuclei may be pretreated prior to in situ reverse transcription.
  • Pretreatment includes fixation and permeabilization with organic solvents.
  • organic solvents are methanol and paraformaldehyde.
  • the working concentration of methanol is 50%-100%, and the working concentration of paraformaldehyde is 1%-4%.
  • Fixation can reduce the free degree and loss of mRNA during reverse transcription.
  • Permeabilization increases the permeability of cells or nuclei. Both fixation and permeabilization can improve the efficiency of reverse transcription and template replacement while ensuring the integrity of cells or nuclei.
  • Pretreatment of cells or nuclei and the inclusion of glycerol in the in situ reverse transcription system also enables simultaneous encapsulation of multiple cells in one droplet in subsequent operations, thereby greatly improving the capture efficiency of the droplet.
  • the method of the present invention comprises the overloading of cells or nuclei after in situ reverse transcription and pretreated magnetic beads.
  • FIG. 4 illustrates droplet generation operations for implementing overloading, according to an embodiment of the invention.
  • Overloading is achieved by controlling the density of cells or nuclei in the single cell suspension to 100-1000 per ⁇ L and the density of magnetic beads to 2000-5000 per ⁇ L.
  • the density of cells or nuclei is 500 per ⁇ L and the density of magnetic beads is 3000 per ⁇ L.
  • the concentration of cells or nuclei is adjusted, so as to be suitable for the subsequent in situ reverse transcription operation and improve the efficiency of in situ reverse transcription. By encapsulating multiple magnetic beads in one droplet at the same time, the utilization efficiency of the magnetic beads is improved.
  • the method of the invention comprises performing a droplet PCR reaction.
  • a PCR reaction system for second-strand cDNA synthesis was added to the droplet PCR reaction.
  • the PCR reaction system for the second-strand cDNA synthesis includes: primers and PCR reagents that are complementary to the 3'-end of the first-strand cDNA and magnetic bead primers (Magnetic Beads Oligo Primer).
  • the primers complementary to the 3'-end of the full-length first-strand cDNA and the magnetic bead primer are Tn primers (Tn Primer).
  • the sequence of the Tn primer is 5'-CGTAGCCATGTCGTTCTG-3'. The Tn primer is consistent with the 5'-end of the magnetic bead sequence and is complementary to the 3'-end of the cDNA template, enabling exponential amplification of the cDNA within the droplet.
  • the magnetic bead is a magnetic bead with a second label
  • the second label includes: Tn primer, second round of label (Round 2 Barcode) and cDNA capture according to the order of the distance from the magnetic bead from near to far.
  • Sequence (Capture oligo), through which the cDNA capture sequence recognizes the first marker on the full-length first-strand cDNA with the first marker generated by in situ reverse transcription to capture the first-strand cDNA.
  • the capture sequence in the second label of the magnetic bead has the following sequence: 5'-TCGTCGGCAGCGTC-3'.
  • the second round of labels includes the first label of the second round (Round 2 Barcode 1) and the second label of the second round (Round 2 Barcode 2).
  • the second round of tags includes a second round of first tags, a second round of second tags and a first linker (linker) between them.
  • the sequence of the first linker is: 5'-CCTTCC-3'. The first linker is designed to connect the second-round first tag and the second-round second tag during the preparation of magnetic beads, and it does not participate in capturing cDNA.
  • a second linker is also included between the Tn primer and the magnetic bead.
  • the second linker is 5 T bases labeled with biotin at the 5'-end, and the sequence is: 5'-biotin-TTTTTT-3'.
  • Biotin can specifically bind to the streptavidin on the magnetic beads, so the second linker can connect the oligonucleotide and the magnetic beads.
  • the PCR amplification of cDNA is completed in the droplet through the droplet PCR reaction, so that multiple magnetic beads of the same droplet can fully capture the cDNA. Since each cDNA molecule carries a different single-molecule marker, multiple magnetic beads of the same droplet can be combined according to the correlation of their captured single-molecule markers, enabling the differentiation of magnetic beads from the same droplet.
  • the method of the present invention also performs fragmentation library construction on the secondary amplified cDNA obtained in step 4).
  • fragmentation library construction a chain cDNA library is obtained, and the chain cDNA library is a single-cell transcriptome sequencing library.
  • the chain cDNA library is further prepared into a circular cDNA library, and the circular cDNA library is a single-cell transcriptome sequencing library.
  • preparing the chained cDNA library into a circular cDNA library includes: melting the double-stranded cDNA in the chained cDNA library under the action of a circularization auxiliary sequence to obtain a melting product; using DNA ligase The melted products were ligated to obtain single-stranded circularized products; the single-stranded circularized products were degraded by enzyme digestion to degrade the remaining uncircularized single-stranded cDNA and double-stranded cDNA to obtain a circularized cDNA library.
  • the degraded single-stranded circularized product is further purified to obtain a circularized cDNA library.
  • magnetic beads are used to purify the single-stranded circularized product to obtain a circularized cDNA library.
  • a single-cell transcriptome sequencing library is constructed as above, and then the single-cell transcriptome sequencing library is sequenced on a machine.
  • the single-cell transcriptome sequencing kit includes at least one of the following items: a tag-containing oligo-dT primer with a first label, a template switching primer, reverse transcriptase, glycerol, immobilized And/or permeabilization reagents, primers complementary to the 3'-end of the first-strand cDNA and magnetic bead primers, magnetic beads with a second label, stranded cDNA library construction reagents, and circular cDNA library construction reagents.
  • the oligo-dT primer comprises in order from 5' to 3': a. a magnetic bead capture region, which is a complementary region to the oligonucleotide in the second label on the magnetic bead ; b. single-molecule markers for identifying individual cDNA transcripts; c. first-round labeling for distinguishing between different cells or nuclei; and d. poly-T sequences for capturing mRNA;
  • the 5'-end of the product includes, in order from 5' to 3': magnetic bead capture region, which is a complementary region to the oligonucleotide in the second label on the magnetic bead; single-molecule labeling; first round Tags and poly-T sequences.
  • the template switching primer is designed to contain cytosine, preferably, the number of cytosine is 3.
  • the reverse transcriptase is a reverse transcriptase with terminal transferase activity, more preferably a reverse transcriptase with terminal transferase activity selected from Maxima H Minus reverse transcriptase (ThermoFisher, EP0752), SuperScript IV reverse transcriptase (ThermoFisher, EP0752, 18090010) or SuperScript TM II reverse transcriptase (ThermoFisher, 18064022).
  • the final concentration of glycerol in the in situ reverse transcription system is 1%-10%.
  • the fixation and/or permeabilization solvent is methanol or paraformaldehyde. In one embodiment, the working concentration of methanol is 30%-100%, and the working concentration of paraformaldehyde is 0.1%-4%.
  • the second label in the magnetic beads with the second label, includes in order of distance from the magnetic bead from near to far: Tn primers, a second round of labels, and a marker for capturing the first-strand cDNA.
  • cDNA capture sequence more preferably, the second round of labeling includes the second round of the first label and the second round of the second label, and further preferably, the second round of the first label and the second round of the second label also includes the second round of the second label
  • a linker more preferably, a second linker is also included between the Tn primer and the magnetic beads.
  • FIG. 5 shows the results of ultra-high-throughput single-cell transcriptome sequencing using the human 293T cell line according to an embodiment of the present invention.
  • the sample is methanol-fixed human 293T cell line, and the first round of labeling in this experiment uses 24 kinds of labels.
  • nearly 20,000 usable cells were obtained from a single chip.
  • FIG. 6 shows the results of ultra-high-throughput single-cell transcriptome sequencing using human 293T and murine 3T3 mixed cell lines according to an embodiment of the present invention.
  • the sample is methanol-fixed human 293T and mouse 3T3 mixed cell line, and the first round of labeling in this experiment uses 48 kinds of labels.
  • about 30,000 usable cells were obtained from a single chip.
  • the double-pack rate of human and mouse cells was 1.59%.
  • FIG. 7 shows the results of ultra-high-throughput single-cell transcriptome sequencing using human 293T and murine 3T3 mixed cell lines according to an embodiment of the present invention.
  • the sample is methanol-fixed human 293T and mouse 3T3 mixed cell line, and the first round of labeling in this experiment uses 96 kinds of labels.
  • 80,000 usable cells were obtained from a single chip.
  • the double-pack rate of human and mouse cells is 10.65%.
  • the obtained cell line or solid tissue For the obtained cell line or solid tissue, prepare a suspension of single cells or nuclei with a suitable digestion method and/or grinding method, and wash the prepared suspension of single cells or nuclei with PBS solution containing 0.04% BSA 1- 2 times and filtered using a 40 ⁇ m cell sieve (BD, 352340). Single cells or nuclei are then fixed, for example, with ice-cold methanol for 20 minutes. The fixed single cells or nuclei were washed 1-2 times with PBS solution containing 0.04% BSA, and filtered using a 40 ⁇ m cell mesh. Then centrifuge at 300-500g and 4°C for 5 minutes to collect the pellet of cells or nuclei. Then add 100 ⁇ L of cell resuspension buffer to resuspend the cells or nuclei, and use a cell counter or counter to detect the concentration of cells/nuclei to 5,000 cells/ ⁇ L.
  • PBS solution containing 0.04% BSA 1- 2 times filtered using
  • the tagged oligo-dT primer sequence is: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACNNNNNNNNNNNNNNJJJJJJJJVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • N is 4 kinds of random bases, a total of 13, this section is a single molecular marker (UMI); J is a designed 96-384 kinds of tags (barcode), a total of 10 bases; then 30 T ; V represents 3 kinds of bases except T; the last one is a random base N.
  • UMI single molecular marker
  • J is a designed 96-384 kinds of tags (barcode), a total of 10 bases; then 30 T ; V represents 3 kinds of bases except T; the last one is a random base N.
  • a suspension of single cells or nuclei was added in an amount of 10,000 cells or nuclei/well (ie, 2 ⁇ L of suspension/well). The system was subsequently incubated at 55°C for 5 minutes to unravel the RNA secondary structure, and then placed immediately on ice to prevent its recombination.
  • this reverse transcription mixed solution contains: 1 ⁇ reverse transcription buffer, 0.5mM dNTP, 2U/ ⁇ L RNase inhibitor, 10U/ ⁇ L Maxima H enzyme (Thermo Scientific, EP0751), 10 % glycerol and 2.5 ⁇ M Tn-TSO (5'-CGTAGCCATGTCGTTCTG rGrG+G -3', where "+G” represents locked nucleotide modified guanine, and "rG” is guanine nucleotide).
  • Cells or nuclei are recovered after in situ reverse transcription. Add 40 ⁇ L of washing buffer to each well, which is a PBS solution containing 0.04% BSA, then recover the treated cells or nuclei, and combine the liquid after several washes in a 1.5 mL centrifuge tube (Invitrogen, AM12400 ) for maximum recovery. Centrifuge at 500rcf, 4°C for 10 minutes to collect cells or nuclei, and wash with 3mL washing buffer repeatedly once. Resuspend the resulting pellet in 50 ⁇ L of wash buffer. Take 2 ⁇ L for dilution and count.
  • washing buffer which is a PBS solution containing 0.04% BSA
  • the PCR premix solution used here contained: 1 ⁇ Fidelity buffer 20 ⁇ L (KAPABIOSYSTEMS, KK2103); 0.3mM dNTP mixture 3 ⁇ L, 3.75mM MgCl 2 7 ⁇ L, 0.2uM Tn primer, 10% Opti-prep 16.7 ⁇ L (Sigma, D1556 -250ML), 1U/ ⁇ L KAPA polymerase 8 ⁇ L (add separately when running the droplet); finally, supplement the total volume to 100 ⁇ L with H 2 O. Due to the high density of Opti-prep, magnetic beads suspended in it will not affect the density due to sedimentation, so it can be used to suspend magnetic beads.
  • the marker sequence is: 5'-TTTTTCCCGTAGCCATGTCGTTCTGCGJJJJJJCCTTCCJJJJJJJTCGTCGGCAGCGTC-3', wherein, the J base is the magnetic bead label (barcode) sequence, a total of 1536 species, each with two segments, after random combination, A total of 1536*1536 kinds of magnetic beads) into magnetic beads (300,000 pieces) in a 0.2mL PCR tube (Axygen, 14-222-260), placed on a magnetic stand for 2 minutes, and discarded the supernatant. Remove the PCR tube from the magnetic stand, add 200 ⁇ L 1 ⁇ buffer D, suspend the magnetic beads, and incubate at room temperature for 5 minutes. Buffer D contains: 1 mM EDTA and 9 mg/mL 85% KOH.
  • LSWB buffer contained: 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 and H2O .
  • the PCR master mix solution used here contains: 0.02% SDS 40 ⁇ L, 1 ⁇ Fidelity buffer 20 ⁇ L, 0.3 mM dNTP mix 3 ⁇ L, 3.75 mM MgCl 2 7 ⁇ L, 0.2 uM Tn primer, 10% Opti-prep 16.7 ⁇ L; and H 2 O 11.3 ⁇ L.
  • Droplet generation is required prior to the droplet PCR reaction. Tear off the surface protective film of the chip (manufacturer: Hanguang; model: Dow Corning 184, 110-1), and place it in the chip groove area of the droplet generating device; connect the A end of the connecting tube on the collection cover (which is in contact with the collection tube) Connecting tube at the bottom) into the outlet hole of the chip; place a 50mL syringe (BD, 300136) on the holder, and adjust the push rod to the initial position. Use a flat needle to connect the syringe to the connection tube B end on the collection tube cap (which is the connection tube that does not touch the bottom of the collection tube). Subsequently, 200 ⁇ L of droplet generating oil (BIO-RAD, 1863005) was added to the collection tube, the collection cap was tightened, and the collection tube was placed vertically on a fixed rack.
  • BIO-RAD droplet generating oil
  • the adapter ligation product was purified using 1.0x Novozyme DNA purification magnetic beads (Vazyme, #N411), and the concentration of the ligation product was determined with qubitdsDNA High Sensitivity Detection Kit (Thermo, Q33230).
  • Primer F 5'-PhoCGTAGCCATGTCGTTCTG*C*-3', where PhoC at the 5'-end is phosphorylated C, and the two bases G*C* at the 3'-end are thio-modified.
  • R primer 5'-TGTGAGCCAAGGAGTTGTTGTCTTTCCTAAGACCGCT-3'.
  • Adapter primer PCR amplification was performed according to the following scheme:
  • the product was purified using 0.6x+0.6x Novizym DNA purification magnetic beads (Vazyme, #N411), and the product was quantified using the qubit dsDNA High Sensitivity Detection Kit (Thermo, Q33230).
  • the obtained cDNA products need to be denatured before sequencing. Take 200-400ng of the cDNA product obtained above, supplement the volume with NF-H 2 O to 47 ⁇ L, add 3 ⁇ L of 20 uM splint oligonucleotide (splint oligo) (5'-GACATGGCTACGTGTGAGCCAAGG-3'), briefly vortex to mix evenly, Centrifuge briefly for 5 seconds. Afterwards, place the PCR tube on the PCR instrument, set the heat lid to 105°C, heat at 95°C for 3 minutes, and then quickly place it on ice for 5-10 minutes.
  • splint oligo 20 uM splint oligonucleotide
  • stop solution 0.1M EDTA
  • ultra-high-throughput single-cell transcriptome sequencing was performed using the human 293T cell line.
  • 24 tags were used in the first round of labeling. Reads in cells: 0.769, average number of single-molecule markers per cell: 1008, average genes per cell: 800. In the end, nearly 20,000 usable cells were obtained from a single chip.
  • Example 3 Ultra-high-throughput single-cell transcriptome sequencing using human 293T and murine 3T3 mixed cell lines
  • ultra-high-throughput single-cell transcriptome sequencing was performed using human 293T and mouse 3T3 mixed cell lines. Using methanol to fix the human 293T and murine 3T3 mixed cell line, 48 tags were used in the first round of labeling. Reads in cells: 0.626, average number of single-molecule markers per cell: 1127, average genes per cell: 469. In the end, about 30,000 usable cells were obtained from a single chip. The double-pack rate of human and mouse cells was 1.59%.
  • Example 4 Ultra-high-throughput single-cell transcriptome sequencing using human 293T and murine 3T3 mixed cell lines
  • ultra-high-throughput single-cell transcriptome sequencing was performed using human 293T and mouse 3T3 mixed cell lines. Using methanol to fix the mixed human 293T and murine 3T3 cell line, 96 tags were used in the first round of labeling. Reads in cells: 0.766, average number of single-molecule markers per cell: 661, average genes per cell: 104. In the end, about 80,000 usable cells were obtained from a single chip. The double-pack rate of human and mouse cells is 10.65%.
  • the method of the present invention realizes ultra-high-throughput single-cell transcriptome sequencing of cells derived from different cell lines and tissues, specifically:
  • the method of the present invention realizes the overloading of cells or nuclei after in situ reverse transcription and pretreated magnetic beads, and provides an efficient, flexible and easy-to-use large-scale scRNA sequencing method.
  • First-round labeling can further expand throughput by adding first-round labeling species. Since each well receives a unique barcode in the first round of labeling, hundreds or even thousands of individual samples can be multiplexed directly in a single experiment—for example, measuring the impact of transcriptomes on large numbers of genetic or pharmacological studies in cell lines perturbation, or processing tissue samples from large epidemiological cohorts.
  • the method of the present invention is simple in operation and high in efficiency, and realizes ultra-high throughput at the level of 100,000 cells in a single experiment.
  • the present method combines the scalability of combinatorial indexing with the efficiency and ease-of-use of droplet generators in a simple two-stage workflow.
  • the present invention demonstrates a 10-fold increase in the throughput of a single experiment compared to a common droplet-seq experiment.
  • the experimental equipment required by the invention is simple, only standard laboratory equipment and a microfluidic droplet generator are required, and only two rounds of indexing are required to complete reverse transcription in intact cells or nuclei.

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Abstract

提供了一种单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒。所述方法中，对单细胞悬液进行细胞或细胞核内原位逆转录，之后将该细胞或细胞核与磁珠超载到液滴中进行液滴PCR反应，之后回收所述磁珠并进行二次扩增、片段化文库构建，从而得到所述单细胞转录组测序文库。所述方法实现了经原位逆转录后的细胞或细胞核与经预处理的磁珠的超载，实现了单次实验获得十万个细胞级别的超高通量，并且表现出相当高的准确性。

Description

单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒 技术领域
本发明涉及分子生物学技术领域，具体而言，涉及一种单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒。
背景技术
单个细胞是生物系统结构和功能的基本单位。然而，常规的基于大量细胞用于分析生物样品分子含量的方法掩盖了生物系统中细胞的异质性。2009年出现第一项单细胞测序技术，之后越来越多的研究基于单细胞的水平，分析单个细胞的转录组已成为解决生物异质性的有效策略。
由于细胞中mRNA种类的表达水平与细胞功能有关，因此单细胞转录组测序可以用来分类细胞类型和检测细胞状态。尽管单细胞RNA测序(sc-RNA-seq)的方法已经激增，但传统的液滴微流控方法获得单细胞RNA测序数据很昂贵，其成本随着处理的细胞数量呈线性增长。此外，由于需要避免细胞在液滴中的双包情况，需要将细胞的密度调整到很低的浓度，才能去装载液滴。这样就导致了单次实验捕获细胞的效率极低，无形中又增加了单细胞RNA测序的成本。
对细胞提前进行标记是一种非常好的策略，能够用于区分同一个液滴中的细胞。并且目前已经有一些方法能够实现对细胞内的RNA提前标记。最常用的方法就是在细胞核内进行RNA的逆转录，用带有标记的寡聚胸腺嘧啶(寡聚-dT)引物与mRNA的多聚腺苷酸(多聚A)尾巴首先进行杂交，再在逆转录酶的作用下以RNA为模板逆转录成cDNA。这样所获得的cDNA产物就携带了寡聚-dT引物上的标签，通过这种方法实现对细胞内转录本的提前标记。
提前标记的方法需要实现在细胞或细胞核水平的逆转录。目前已经有一些技术用到了这个方法。Cao et al.等发表的文章(Comprehensive si ngle-cell transcriptional profili ng of a m μLticell μLar organism；Science；18Aug 2017：Vol.357，Issue 6352，pp.661-667)公开了sci-RNA-seq技术以及sci-RNA-seq3技术。在两项技术中，首先将细胞平均分到96或384孔板中，每个孔中加入一种带有标记的寡聚-dT引物，也就是说，第一轮标记一共有96或者384种标记。然后每孔加入同样的逆转录试剂，进行逆转录反应。第一轮标记结束后，回收孔板内所有的细胞或细胞核。再将回收产物平均分到第二个96或384孔板中，同样的，每孔加入一种不同标记的引物，也就是说，第二轮标记同样一共有96或者384种标记。在第二轮中，通过PCR(sci-RNA-seq)或连接反应(sci-RNA-seq3)加上标记。这种通过多轮细胞标记的方法在单次实验中能够获得上万个细胞，因此极大提高了单次实验的通量，从而节省了成本。然而，这种实验方法的缺点在于其是基于孔板进行的手工操作，无法实现自动化细胞捕获， 大大增加了工作量；此外，其实验步骤非常繁琐；并且每轮细胞回收都会有一定的损失，导致细胞的回收率较低。
用于提前标记的另一种方法是基于液滴微流控的单细胞测序技术(drop-seq)。Macosko et al.等发表的文章(Highly Parallel Genome-wide Expression Profili ng of Individual Cells Usi ng Nanoliter Droplets；Cell；May 21，2015，Vol.161，Issue 5，pp.1202-1214)公开了一种使用μL液滴的单细胞测序方法。在这种方法中，基于液滴微流控技术采用油包水的液滴对细胞和微珠(micro-particle)进行包裹，得到同时含有一个细胞和一个微珠的液滴。液滴内其他成分还包括裂解液，并且微珠上含有大量被称为标签(barcode)的带有特定分子标记的DNA序列。在液滴不断生成过程中，液滴内的细胞受到裂解液作用，释放出大量mRNA，与此同时，mRNA能被微珠捕获。待全部液滴生成完成以后，滴液被打破。之后，mRNA被逆转录成cDNA，并进一步被扩增。然后，通过打断酶处理cDNA使其断裂，并在两端加上测序接头，随后对加上接头的cDNA进行测序。但是这种方法中，为了使双微珠或者双包出现概率最小化，必须将微珠相和细胞相的密度降低到很低的浓度。因此，这种方法难以实现超过一万个细胞/次实验的超高通量单细胞测序，无形中增加了每个细胞的成本。
因此，对传统的液滴微流控方法以及细胞提前标记方法进行改造以提高效率是亟待解决的问题。
发明内容
本发明的主要目的在于提供一种单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒以解决现有技术中单细胞转录组测序文库构建效率低的问题。
为了实现上述目的，根据本发明的第一方面，本发明提出了一种单细胞转录组测序文库的构建方法。方法包括以下步骤：
1)：对单细胞悬液进行细胞或细胞核内原位逆转录以产生带有第一标记的全长第一链cDNA；2)：将步骤1)获得的经原位逆转录后的细胞或细胞核和用于第二链cDNA合成的PCR反应体系与能够识别第一标记的磁珠超载到液滴中，进行液滴PCR反应；3)：在液滴PCR反应结束后回收磁珠；4)：对在步骤3)中获得的磁珠上的cDNA进行二次扩增；和5)：对在步骤4)中获得的经二次扩增后的cDNA进行片段化文库构建，得到单细胞转录组测序文库。
进一步地，在步骤1)中，采用含标签的寡聚-dT引物进行原位逆转录从而产生带有第一标记的全长第一链cDNA；其中，寡聚-dT引物按照从5’到3’的顺序依次包括：a.磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；b.用于识别单个cDNA转录本的单分子标记；c.用于区分不同细胞或细胞核的第一轮标签；和d.用于捕获mRNA的多聚T序列；从而使得在所获得的具有第一标记的全长第一链cDNA的5’-端按照从5’到3’的顺序依次包括：磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；单分子标记；第一轮标签和多聚T序列，其中，磁珠能够识别第一标记中的磁珠捕获区。
进一步地，在步骤1)中，采用具有末端转移酶活性的逆转录酶进行原位逆转录，并且原位逆转录的体系中还包含模板置换引物(Tn-TSO)，优选地，具有末端转移酶活性的逆转录酶选自Maxima H酶、SSII酶或SSIV酶；优选地，模板置换引物经设计包含位于3’-端的鸟嘌呤，优选地，鸟嘌呤的个数为3个。
进一步地，原位逆转录的体系中还包含甘油，优选地，甘油在原位逆转录的体系中的终浓度为5％～10％；优选地，构建方法还包括在进行原位逆转录前对单细胞悬液中的细胞或细胞核进行固定和/或透化预处理；更优选地，使用有机溶剂进行固定和/或透化预处理；进一步优选，有机溶剂为甲醇或多聚甲醛；更进一步优选地，甲醇的工作浓度为50％～100％，多聚甲醛的工作浓度为1％～4％。
进一步地，在步骤2)中，通过控制单细胞悬液中的细胞或细胞核的密度为100～1000个每μL，并且磁珠的密度为2000～5000个每μL；优选地，细胞或细胞核的密度为500个每μL，并且磁珠的密度为3000个每μL。
进一步地，步骤2)中的用于第二链cDNA合成的PCR反应体系包括：同时与第一链cDNA的3’-端以及磁珠引物互补的引物以及PCR试剂；优选地，同时与全长第一链cDNA的3’-端以及磁珠引物互补的引物为Tn引物。
进一步地，步骤2)中的磁珠为带有第二标记的磁珠，优选地，第二标记按照与磁珠距离由近及远的顺序依次包括：Tn引物、第二轮标签以及用于识别并捕获全长第一链cDNA的cDNA捕获序列；更优选地，第二轮标签包括第二轮第一标签和第二轮第二标签；进一步优选地，第二轮第一标签和第二轮第二标签之间还包括第一接头；更进一步优选地，Tn引物与磁珠之间还包括第二接头。
进一步地，步骤5)包括：将步骤4)中获得的经二次扩增后的cDNA进行片段化文库构建，得到链状cDNA文库，链状cDNA文库即为单细胞转录组测序文库；优选地，进一步将链状cDNA文库制备成环状cDNA文库，环状cDNA文库即为单细胞转录组测序文库；优选地，将链状cDNA文库制备成环状cDNA文库包括：将链状cDNA文库中的双链cDNA在环化辅助序列的作用下进行解链，得到解链产物；采用DNA连接酶对解链产物进行连接，得到单链环化产物；对单链环化产物进行酶切降解残留未环化的单链cDNA和双链cDNA，得到环化cDNA文库；更优选地，在对单链环化产物进行酶切降解之后，进一步对降解后的单链环化产物进行纯化，得到环化cDNA文库；进一步优选，采用磁珠对单链环化产物进行纯化，得到环化cDNA文库。
根据本发明的第二方面，本发明提出了一种单细胞转录组文库的测序方法，其特征在于，测序方法包括：
按照本发明的第一方面的构建方法构建单细胞转录组测序文库；和将单细胞转录组测序文库进行上机测序。
根据本发明的第三方面，本发明提出了一种制备单细胞转录组文库的试剂盒，其特征在于，试剂盒包括以下各项中至少一种：带有第一标记的含标签的寡聚-dT引物、模板转换引物、逆转录酶、甘油、固定和/或透化试剂、同时与全长第一链cDNA的3’-端以及磁珠引物互补的引物、带有第二标记的磁珠、链状cDNA文库构建试剂和环状cDNA文库构建试剂；优选地，含标签的寡聚-dT引物按照从5’到3’的顺序依次包括：a.磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；b.用于识别单个cDNA转录本的单分子标记；c.用于区分不同细胞或细胞核的第一轮标签；和d.用于捕获mRNA的多聚T序列；优选地，逆转录酶为具有末端转移酶活性的逆转录酶，更优选具有末端转移酶活性的逆转录酶选自Maxima H酶、SSII酶或SSIV酶；优选地，模板转换引物经设计包含鸟嘌呤，优选地，鸟嘌呤的个数为3个；优选地，甘油在原位逆转录的体系中的终浓度为5％～10％；优选地，固定和/或透化溶剂为有机溶剂，更优选地，有机溶剂为甲醇或多聚甲醛，更进一步优选地，甲醇的工作浓度为50％～100％，多聚甲醛的工作浓度为1％～4％；优选地，同时与全长第一链cDNA的3’-端以及磁珠引物互补的引物为Tn引物；优选地，带有第二标记的磁珠中，第二标记按照与磁珠的距离由近及远的顺序依次包括：Tn引物、第二轮标签以及用于捕获第一链cDNA的cDNA捕获序列，更优选地，第二轮标签包括第二轮第一标签和第二轮第二标签，进一步优选地，第二轮第一标签和第二轮第二标签之间还包括第一接头，更进一步优选地，Tn引物与磁珠之间还包括第二接头；优选地，链状cDNA文库构建试剂用于逆转录及扩增后的cDNA进行片段化文库构建，得到链状cDNA文库；优选地，环状cDNA文库构建试剂用于将链状cDNA文库制备成环状cDNA文库。
应用本发明的技术方案，实现了经原位逆转录后的细胞或细胞核与经预处理的磁珠的超载，提供了一种高效、灵活且易于使用的大规模scRNA测序方法。本发明方法操作简单，效率高，可以高效构建单细胞转录组测序文库，从而大大提高了基于液滴微流控的单细胞转录组测序方法的通量，实现单次实验获得十万个细胞级别。此外，本发明在实验上节约了成本，因而有利于大规模单细胞研究项目的开展。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了现有技术的sci-RNA-seq技术的流程图。在该技术中，首先将细胞平均分到96孔板中，每个孔中加入一种带有标记的寡聚-dT引物，也就是说，第一轮标记一共有96种标记。然后每孔加入同样的逆转录试剂，进行逆转录反应。第一轮标记结束后，回收孔板内所有的细胞或细胞核。再将回收产物平均分到第二个96孔板中，同样的，每孔加入一种不同标记的引物。第二轮标记同样一共有96或者384种标记。第二轮通过PCR反应加上标记。
图2示出了现有技术的基于液滴微流控的单细胞测序技术的流程图。在该技术中，首先采用油包水的液滴对细胞和微珠进行包裹，得到同时含有一个细胞和一个微珠的液滴，液滴内其他成分还包括裂解液，并且微珠上含有大量被称为标签的带有特定分子标记的DNA序列。 在液滴不断生成过程中，液滴内的细胞被裂解液裂开，释放出大量mRNA，与此同时，mRNA能被微珠捕获。待全部液滴生成完成以后，滴液被打破。mRNA被逆转录成cDNA，进一步被扩增。然后，cDNA被打断酶打断并且两端加上测序接头。进一步，加上接头的cDNA进行测序。
图3示出了根据本发明的单细胞转录组测序文库的构建方法以及单细胞转录组文库的测序方法的流程图。首先，对单细胞悬液进行细胞或细胞核内原位逆转录以产生带有第一标记的全长第一链cDNA；然后将获得的经原位逆转录后的细胞或细胞核和用于第二链cDNA合成的PCR反应体系与能够识别第一标记的磁珠超载到液滴中，进行液滴PCR反应；然后在液滴PCR反应结束后回收磁珠；对磁珠上的cDNA进行二次扩增；和经二次扩增后的cDNA进行片段化文库构建，得到单细胞转录组测序文库。在获得单细胞转录组测序文库进行上机测序。
图4示出了根据本发明的实施方式的用于实现超载的液滴生成操作。将芯片表面保护膜撕掉，放置于液滴发生装置的芯片槽区域；将收集盖子上的连接管A端(其是接触收集管底部的连接管)插入芯片的出口孔；将50mL注射器(BD，300136)放置在固定架，并调节推杆到初始位置。用平口针头连接注射器和收集管盖子上的连接管B端(其是非接触收集管底部的连接管)。随后向收集管加入200μL液滴生成油，旋紧收集盖子，并将收集管竖直放置于固定架上。使用移液器轻轻吹打混匀细胞或细胞核的悬液，随后向芯片的细胞孔加入100μL细胞或细胞核的悬液，确保枪头接触孔底部。使用移液器轻轻吹打混匀磁珠，向芯片的磁珠孔加入100μL吹打混匀的磁珠，确保枪头接触孔底。立即添加350μL液滴生成油到芯片的油孔，迅速将注射器的推杆拉到卡槽位置，将推杆卡在卡槽处，启动计时器，5分钟后开始收集液滴，立即拧松收集管上的收集盖子，拔出芯片出口孔的连接管，竖直拉伸连接管，让管中的液滴流入收集管中，然后换上普通的收集管盖子。
图5示出了根据本发明的实施方式的使用人源293T细胞系的超高通量单细胞转录组测序的结果。在该图中使用样本为甲醇固定的人源293T细胞系，本次实验第一轮标记采用24种标签。最终单张芯片得到将近2万个可用细胞。
图6示出了根据本发明的实施方式的使用人源293T和鼠源3T3混合细胞系的超高通量单细胞转录组测序的结果。在该图中使用样本为甲醇固定的人源293T和鼠源3T3混合细胞系，本次实验第一轮标记采用48种标签。最终单张芯片得到约3万个可用细胞。人鼠细胞双包率为1.59％。
图7示出了根据本发明的实施方式的使用人源293T和鼠源3T3混合细胞系的超高通量单细胞转录组测序的结果。在该图中使用样本为甲醇固定的人源293T和鼠源3T3混合细胞系，本次实验第一轮标记采用96种标签。最终单张芯片得到8万个可用细胞。人鼠细胞双包率为10.65％。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
如背景技术部分所描述的，常规的基于大量细胞用于分析生物样品分子含量的方法掩盖了生物系统中细胞的异质性。因此，越来越多的研究基于单细胞的水平，分析单个细胞的转录组已成为解决生物异质性的有效策略。尽管单细胞RNA测序(sc-RNA-seq)的方法已经激增，但传统的方法仍存在较多问题。
图1示出了现有技术的sci-RNA-seq技术的流程图。在该传统的多轮细胞标记的方法中，虽然在单次实验中能够获得上万个细胞，因此极大提高了单次实验的通量，从而节省了成本，然而，这种实验方法的缺点在于其是基于孔板进行的手工操作，无法实现自动化细胞捕获，大大增加了工作量；此外，其实验步骤非常繁琐；并且每轮细胞回收都会有一定的损失，导致细胞的回收率较低。
图2示出了现有技术的基于液滴微流控的单细胞测序技术的流程图。在这种传统的基于液滴微流控的单细胞测序技术中，为了使双微粒或者双包出现概率最小化，必须将微粒相和细胞相的密度降低到很低的浓度。因此，这种方法难以实现超过一万个细胞/次实验的超高通量单细胞测序，无形中增加了每个细胞的成本。
图3示出了根据本发明的单细胞转录组测序文库的构建方法以及单细胞转录组文库的测序方法的流程图。为了对传统的液滴微流控方法以及细胞提前标记方法进行改造，本发明提供一种单细胞转录组测序文库的构建方法以及基于这种单细胞转录组测序文库的构建方法的单细胞转录组文库的测序方法。
根据本发明的实施方式的单细胞转录组测序文库的构建方法首先在孔板中对单细胞悬液进行细胞或细胞核内原位逆转录以产生带有第一标记的全长第一链cDNA，从而通过原位逆转录对细胞或细胞核进行提前标记。这一步所有细胞的标记种类从24种到384种不等。随后，将经原位逆转录后的细胞或细胞核和用于第二链cDNA合成的PCR反应体系与磁珠超载到液滴中。通常，液滴有1～5个细胞或细胞核，相应地，保证该液滴中有1～10个磁珠。通过超载使几乎所有液滴中都有磁珠并且有效细胞或细胞核的包裹率也大大提高。在使细胞或细胞核与磁珠超载的同时装载用于第二链cDNA合成的PCR反应体系，进行液滴PCR反应，使液滴中的磁珠能够均匀捕获经原位逆转录获得的cDNA。之后对cDNA进行二次扩增并构建cDNA文库。
根据本发明的实施方式的单细胞转录组文库的测序方法首先构建单细胞转录组测序文库，并将单细胞转录组测序文库进行上机测序。在得到高通量的测序数据后，根据磁珠之间捕获片段的相关性，首先组合出来自同一个液滴的磁珠。在一个液滴中将细胞进行裂解，cDNA释放出来，该液滴中的磁珠随机捕获释放的cDNA。因此，一个液滴中的磁珠所捕获的片段都是该液滴中的细胞的cDNA片段。同时在液滴中通过PCR扩增cDNA模板，完成扩增后，模板的拷贝数增加，并且每条复制后的模板被同一个液滴中不同磁珠捕获的概率相等。因此，同一个液滴中即使有几个不同的磁珠，它们所捕获的片段应该是非常一致的，由此可以通过 计算磁珠捕获片段的相关性而组合这种一致的磁珠。因此，根据本发明的方法实现了磁珠在液滴中的超载，从而以提高液滴的利用效率。随后根据第一轮标签区分同一个液滴中超载的细胞或细胞核，从而在提高细胞密度的情况下，区分出双包情况。在本发明中，双包指一个液滴中由两个及以上的细胞；若无法区分双包，则从测序数据中我们只能得到这两个细胞转录本的加和，无法获得单个细胞的特征。会对单细胞下游分析造成困扰。因此，区分出双包情况是必要的。
在计算相关性的过程中，根据预先标记的细胞标签(cell barcode)将磁珠区分开来，此时一个细胞被分成了几个子细胞(sub-cell)，根据不同子细胞之间捕获的单分子标记(UMI)相关性将它们组合起来。由于在液滴中对单分子标记做了PCR，所以同一个细胞的单分子标记会有多个拷贝，然后多个拷贝被液滴里不同磁珠捕获。如果几个子细胞之间拥有较多相同的单分子标记，则说明它们捕获了同一个细胞。
其后，根据单分子标记的相关性区分出一个液滴中的磁珠，并对一致的磁珠进行组合。首先创建一个子细胞*单分子标记(sub-cells*UMI)的矩阵，将单分子标记数目1的值替换为0(此时，将UMI＝1作为背景)，计算子细胞之间的雅卡尔系数(Jaccard index)，根据雅卡尔系数的knee plot选择组合阈值。如果几个子细胞之间其雅卡尔系数高于此阈值，则将它们组合为一个细胞。
通过提高磁珠相的密度实现磁珠在液滴中的超载。根据泊松分布的概率密度计算公式，在此装置中液滴流速一致的情况下，当液滴大小为110μm时，颗粒的密度如果达到3000个/μL，有约70％的液滴会包裹到该颗粒。该实验所用磁珠密度为3000个/μL，经计算，液滴中包裹1个磁珠的概率是36.7％，包裹两个以上磁珠的概率是30％。当液滴包裹2个及以上磁珠的概率大于5％时，被认为实现了磁珠在液滴的超载。在此使用的泊松分布的概率密度函数为：
Poisson(N(t)＝n)＝((λt) ne -λt)/n！，(λt＞0，n≥0)，
其中，λ为平均每个液滴中的细胞数，t为常数。
根据第一轮标记区分超载细胞时，第一轮所用的寡聚-dT共带有96种至384种标记，相当于第一轮把细胞标记成96种至384种。在包裹细胞的液滴生成过程中，根据泊松分布计算公式，人为控制细胞密度，使每个液滴中不超过10个细胞。相当于从96种至384种细胞中随机抽取10个细胞，那么这10个细胞有很大概率带着第一轮中的10种不同标记。同时，每个液滴中的磁珠均带着不同的标记，作为第二轮标记。测序完成后，可根据两轮标记序列将来自同一个液滴的细胞区分。
在一个实施方式中，单细胞转录组测序文库的构建方法包括以下步骤：
1)对单细胞悬液进行细胞或细胞核内原位逆转录以产生带有第一标记的全长第一链cDNA；
2)将步骤1)获得的经原位逆转录后的细胞或细胞核和用于第二链cDNA合成的PCR反应体系与能够识别所述第一标记的磁珠超载到液滴中，进行液滴PCR反应；
3)在液滴PCR反应结束后回收磁珠；
4)对在步骤3)中获得的磁珠上的cDNA进行二次扩增；和
5)对在步骤4)中获得的经二次扩增后的cDNA进行片段化文库构建，得到单细胞转录组测序文库。
在一个实施方式中，在步骤1)中，采用含标签的寡聚-dT(Indexed oligo-dT)引物进行原位逆转录从而产生带有第一标记的全长第一链cDNA。该含标签的寡聚-dT引物按照从5’到3’的顺序依次包括：
a.磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区(也称为适配子，Adaptor)，用于磁珠捕获cDNA；
b.用于识别单个cDNA转录本的单分子标记(Unique molecular index，UMI)；
c.用于区分不同细胞或细胞核的第一轮标签(Round 1 Barcode)，其给细胞标上一轮标签，用来区分细胞；和
d.用于捕获mRNA的多聚T(polyT)序列(dT30VN)。
由此，通过利用寡聚-dT引物使得在所获得的cDNA产物的5’-端按照从5’到3’的顺序依次包括：磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区(也称为适配子，Adaptor)、单分子标记、第一轮标签和多聚T序列(dT30V)。磁珠能够识别所述第一标记中的所述磁珠捕获区。
采用具有末端转移酶活性的逆转录酶进行所述原位逆转录。优选地，具有末端转移酶活性的逆转录酶选自Maxima H酶、SSII酶或SSIV酶。通过逆转录酶的作用将胞嘧啶(C)加在cDNA第一链的末尾，优选胞嘧啶的个数为3个。此时获得cDNA第一链，其结构可以表示为：磁珠捕获区-单分子标记-第一轮标签-多聚T序列-cDNA-CCC。
在一个实施方式中，原位逆转录的体系中还包含模板置换引物，也称为模板置换寡核苷酸(Template switch oligo，Tn-TSO)。在一个实施方式中，模板置换引物的序列为：5’-CGTAGCCATGTCGTTCTG rGrG+G-3’，其中，“+G”表示锁核苷酸修饰的鸟嘌呤，“rG”是鸟嘌呤核苷酸，如此，模板置换引物经设计包含位于3’-端的鸟嘌呤核苷酸(G)，优选鸟嘌呤核苷酸的个数为3个(Tn-TSO- rGrG+G)。这样，模板置换引物通过其鸟嘌呤与cDNA上的胞嘧啶互补配对，即，在所获得的cDNA产物(cDNA第一链)的3’-端CCC与Tn-TSO-rGrG+G的3’-端rGrG+G的序列互补，使得该模板置换引物继续作为逆转录的模板指导cDNA的合成。
在一个实施方式中，原位逆转录的体系中还包含甘油。优选地，甘油在原位逆转录的体系中的终浓度为5％～10％。甘油能够保护细胞或细胞核的完整性。此外，甘油能够减少细胞之间的粘连，间接提高逆转录的效率。
在一个实施方式中，在进行原位逆转录之前，可以对细胞或细胞核进行预处理。预处理包括用有机溶剂进行固定和透化处理。优选的有机溶剂例如甲醇和多聚甲醛。在一个实施方 式中，甲醇的工作浓度为50％～100％，多聚甲醛的工作浓度为1％～4％。固定能够减少逆转录过程中mRNA的游离程度和损失量。透化可以提高细胞或细胞核的通透性。进行固定和透化处理均能够在保证细胞或细胞核完整度的状态下提高逆转录以及模板置换的效率。
通过对细胞或细胞核进行预处理以及在原位逆转录的体系包含甘油，还使得能够在后续操作中实现在一个液滴中对多个细胞的同时包裹，从而极大提高液滴的捕获效率。
在一个实施方式中，本发明的方法包括经原位逆转录后的细胞或细胞核与经预处理的磁珠的超载。图4示出了根据本发明的实施方式的用于实现超载的液滴生成操作。通过将单细胞悬液中的细胞或细胞核的密度控制为100～1000个每μL并且将磁珠的密度控制为2000～5000个每μL，从而实现超载。在一个优选的实施方式中，细胞或细胞核的密度为500个每μL并且磁珠的密度为3000个每μL。通过该步骤调整细胞或细胞核的浓度，从而适合后续的原位逆转录操作，提高原位逆转录的效率。通过在一个液滴中同时包裹多个磁珠，提高了磁珠的利用效率。
在一个实施方式中，本发明的方法包括进行液滴PCR反应。在该液滴PCR反应中加入了用于第二链cDNA合成的PCR反应体系。在一个实施方式中，用于第二链cDNA合成的PCR反应体系包括：同时与第一链cDNA的3’-端以及磁珠引物(Magnetic Beads Oligo Primer)互补的引物以及PCR试剂。优选地，同时与全长第一链cDNA的3’-端以及磁珠引物互补的引物为Tn引物(Tn Primer)。在一个实施方式中，Tn引物的序列为5’-CGTAGCCATGTCGTTCTG-3’。Tn引物与磁珠序列的5’-端一致，并和cDNA模板3’-端互补，能够实现液滴内cDNA的指数扩增。
在一个实施方式中，磁珠为带有第二标记的磁珠，第二标记按照与磁珠距离由近及远的顺序依次包括：Tn引物、第二轮标签(Round 2 Barcode)以及cDNA捕获序列(Capture oligo)，通过该cDNA捕获序列识别经原位逆转录产生的带有第一标记的全长第一链cDNA上的第一标记而捕获该第一链cDNA。在一个实施方式中，磁珠的第二标记中的捕获序列具有如下序列：5’-TCGTCGGCAGCGTC-3’。在一个实施方式中，第二轮标签包括第二轮第一标签(Round 2 Barcode 1)和第二轮第二标签(Round 2 Barcode 2)。在一个实施方式中，第二轮标签包括第二轮第一标签、第二轮第二标签和它们之间的第一接头(连接序列，linker)。在一个实施方式中，该第一接头的序列为：5’-CCTTCC-3’。在磁珠的制备过程中为了连接第二轮第一标签和第二轮第二标签而设计了该第一接头，其不参与捕获cDNA。在一个实施方式中，Tn引物与磁珠之间还包括第二接头。该第二接头是5’-端生物素标记的5个T碱基，序列为：5’-生物素-TTTTT-3’。生物素可以与磁珠上的链霉亲和素特异结合，所以该第二接头能够连接寡聚核苷酸和磁珠。通过液滴PCR反应在液滴中完成cDNA的PCR扩增，使同一个液滴的多个磁珠充分捕获cDNA。由于每个cDNA分子携带了不同的单分子标记，同一个液滴的多个磁珠可以根据其捕获的单分子标记的相关性进行组合，使得能够区分来自同一个液滴的磁珠。
在一个实施方式中，本发明的方法还将步骤4)中获得的经二次扩增后的cDNA进行片段化文库构建。通过片段化文库构建，得到链状cDNA文库，链状cDNA文库即为单细胞转录 组测序文库。在一个实施方式中，进一步将链状cDNA文库制备成环状cDNA文库，环状cDNA文库即为单细胞转录组测序文库。
在一个实施方式中，将链状cDNA文库制备成环状cDNA文库包括：将链状cDNA文库中的双链cDNA在环化辅助序列的作用下进行解链，得到解链产物；采用DNA连接酶对解链产物进行连接，得到单链环化产物；对单链环化产物进行酶切降解残留未环化的单链cDNA和双链cDNA，得到环化cDNA文库。
在一个实施方式中，在对单链环化产物进行酶切降解之后，进一步对降解后的单链环化产物进行纯化，得到环化cDNA文库。在一个实施方式中，采用磁珠对单链环化产物进行纯化，得到环化cDNA文库。
根据本发明的实施方式的单细胞转录组文库的测序方法首先如上构建单细胞转录组测序文库，并随后将单细胞转录组测序文库进行上机测序。
根据本发明的实施方式的单细胞转录组测序的试剂盒包括以下各项中至少一种：带有第一标记的含标签的寡聚-dT引物、模板转换引物、逆转录酶、甘油、固定和/或透化试剂、与第一链cDNA的3’-端以及磁珠引物互补的引物、带有第二标记的磁珠、链状cDNA文库构建试剂和环状cDNA文库构建试剂。
在一个实施方式中，寡聚-dT引物按照从5’到3’的顺序依次包括：a.磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；b.用于识别单个cDNA转录本的单分子标记；c.用于区分不同细胞或细胞核的第一轮标签；和d.用于捕获mRNA的多聚T序列；从而使得在所获得的cDNA产物的5’-端按照从5’到3’的顺序依次包括：磁珠捕获区，其是与磁珠上第二标记中的寡核苷酸互补的互补区；单分子标记；第一轮标签和多聚T序列。
在一个实施方式中，模板转换引物经设计以包含胞嘧啶，优选地，胞嘧啶的个数为3个。在一个实施方式中，逆转录酶为具有末端转移酶活性的逆转录酶，更优选具有末端转移酶活性的逆转录酶选自Maxima H Minus逆转录酶(ThermoFisher，EP0752)、SuperScript IV逆转录酶(ThermoFisher，EP0752，18090010)或SuperScript TM II逆转录酶(ThermoFisher，18064022)。在一个实施方式中，甘油在原位逆转录的体系中的终浓度为1％～10％。在一个实施方式中，固定和/或透化溶剂为甲醇或多聚甲醛。在一个实施方式中，甲醇的工作浓度为30％～100％，，多聚甲醛的工作浓度为0.1％～4％。
在一个实施方式中，带有第二标记的磁珠中，第二标记按照与磁珠的距离由近及远的顺序依次包括：Tn引物、第二轮标签以及用于捕获第一链cDNA的cDNA捕获序列，更优选地，第二轮标签包括第二轮第一标签和第二轮第二标签，更进一步优选地，第二轮第一标签和第二轮第二标签之间还包括第一接头，更进一步优选地，Tn引物与磁珠之间还包括第二接头。
图5示出了根据本发明的实施方式的使用人源293T细胞系的超高通量单细胞转录组测序的结果。在该图中使用样本为甲醇固定的人源293T细胞系，本次实验第一轮标记采用24种标签。最终单张芯片得到将近2万个可用细胞。
图6示出了根据本发明的实施方式的使用人源293T和鼠源3T3混合细胞系的超高通量单细胞转录组测序的结果。在该图中使用样本为甲醇固定的人源293T和鼠源3T3混合细胞系，本次实验第一轮标记采用48种标签。最终单张芯片得到约3万个可用细胞。人鼠细胞双包率为1.59％。
图7示出了根据本发明的实施方式的使用人源293T和鼠源3T3混合细胞系的超高通量单细胞转录组测序的结果。在该图中使用样本为甲醇固定的人源293T和鼠源3T3混合细胞系，本次实验第一轮标记采用96种标签。最终单张芯片得到8万个可用细胞。人鼠细胞双包率为10.65％。
以下结合具体实施例对本发明作进一步详细描述，这些实施例不能理解为限制本发明所要求保护的范围。
实施例1：材料和方法
准备单细胞悬液
在原位逆转录之前的进行单细胞悬液的准备步骤。
针对所获取的细胞系或实体组织，采用合适的消化法和/或研磨法制备单细胞或细胞核的悬液，用包含0.04％BSA的PBS溶液清洗所制备的单细胞或细胞核的悬液1-2次，并使用40μm的细胞筛(BD，352340)过滤。随后对单细胞或细胞核进行固定，例如，可以使用冰甲醇固定20分钟。用包含0.04％BSA的PBS溶液清洗经固定的单细胞或细胞核1-2次，并使用40μm的细胞筛过滤。随后在300-500g、4℃的条件下离心5分钟收集细胞或细胞核的沉淀。之后加入100μL的细胞重悬缓冲液来重悬细胞或细胞核，并使用细胞计数板或计数仪检测细胞/细胞核的浓度至5,000个/μL。
进行细胞或细胞核内原位逆转录
在原位逆转录之前，在多孔板中准备25μM的含标签的寡聚-dT引物，每孔1μL，并将多孔板保存在-20℃。该含标签的寡聚-dT引物序列为：5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACNNNNNNNNNNNNNJJJJJJJJJJVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’。其中，N为4种随机碱基，共13个，此段是单分子标记(UMI)；J为设计好的96种-384种标签(barcode)，共10个碱基；然后是30个T；V代表除了T之外的3种碱基；最后一个是随机碱基N。
在实验开始时，以10,000个细胞或细胞核/孔(即2μL悬浮液/孔)的量添加单细胞或细胞核的悬浮液。随后在55℃下孵育体系5分钟以解开RNA二级结构，然后立即置于冰上以防止其重组。
向每孔加入逆转录混合溶液16μL，该逆转录混合溶液包含：1×逆转录缓冲液，0.5mM dNTP，2U/μL RNA酶抑制剂，10U/μL Maxima H酶(Thermo Scientific，EP0751)，10％甘油和2.5μM Tn-TSO(5’-CGTAGCCATGTCGTTCTG rGrG+G-3’，其中，“+G”表示锁核苷酸修饰的鸟嘌呤，“rG”是鸟嘌呤核苷酸)。
将热盖设为65℃，根据以下程序进行逆转录：
50℃加热10分钟；
3个循环的以下步骤：
{8℃保持12秒，
15℃保持45秒，
20℃保持45秒，
30℃保持30秒，
42℃保持2分钟，和
50℃保持3分钟；}
50℃加热15分钟；
4℃保存。
回收细胞或细胞核
在原位逆转录后回收细胞或细胞核。向每个孔加入40μL洗涤缓冲液，该洗涤缓冲液是包含0.04％BSA的PBS溶液，然后回收经处理的细胞或细胞核，将几次洗涤后的液体合并在一个1.5mL离心管(Invitrogen，AMl2400)中以实现最大回收率。在500rcf、4℃的条件下离心10分钟收集细胞或细胞核，用3mL洗涤缓冲液重复清洗1次。将所得的沉淀重悬于50μL洗涤缓冲液中。取2μL进行稀释后计数。
之后将50,000个细胞加入PCR预混溶液中至总体积100μL，备用。此处使用的PCR预混溶液包含：1×Fidelity缓冲液20μL(KAPABIOSYSTEMS，KK2103)；0.3mM dNTP混合物3μL，3.75mM MgCl 2 7μL，0.2uM Tn引物，10％Opti-prep16.7μL(Sigma，D1556-250ML)，1U/μL KAPA聚合酶8μL(跑液滴时单独添加)；最后，用H 2O将总体积补充至100μL。由于Opti-prep密度较大，磁珠悬浮在其中不会因为沉降而影响密度，因此可用其悬浮磁珠。
磁珠的预处理
对于每次实验，吸取100μL带有标记(标记序列为：5’-TTTTTCCCGTAGCCATGTCGTTCTGCGJJJJJJJJJJCCTTCCJJJJJJJJJJTCGTCGGCAGCGTC-3’，其中，J碱基为磁珠标签(barcode)序列，共1536种，分别有两段，随机组合后，共有1536*1536种磁珠)到0.2mL PCR管(Axygen，14-222-260)的磁珠(300,000个)中，放在磁力架上静置2分钟，弃去上清。从磁力架取下PCR管，加入200μL 1×缓冲液D，悬浮磁珠，室温孵育5分钟。缓冲液D包含：1mM EDTA和9mg/mL 85％KOH。
将PCR管放在磁力架上静置2分钟，弃去上清。保持PCR管在磁力架上，加入200μL 1×缓冲液D，静置30秒弃去上清。加入200μL LSWB缓冲液，静置30秒，然后弃去上清。重复上一步操作。LSWB缓冲液包含：50mM Tris-HCl，150mM NaCl，0.05％吐温-20和H 2O。
随后加入100μL的PCR预混溶液重悬磁珠，备用。此处使用的PCR预混溶液包含：0.02％SDS40μL，1×Fidelity缓冲液20μL，0.3mM dNTP混合物3μL，3.75mM MgCl 2 7μL，0.2uM Tn引物，10％Opti-prep 16.7μL；和H 2O 11.3μL。
生成液滴
在液滴PCR反应之前需要生成液滴。将芯片(厂商：含光；型号：道康宁184型、110-1)表面保护膜撕掉，放置于液滴发生装置的芯片槽区域；将收集盖子上的连接管A端(其是接触收集管底部的连接管)插入芯片的出口孔；将50mL注射器(BD，300136)放置在固定架，并调节推杆到初始位置。用平口针头连接注射器和收集管盖子上的连接管B端(其是非接触收集管底部的连接管)。随后向收集管加入200μL液滴生成油(BIO-RAD，1863005)，旋紧收集盖子，并将收集管竖直放置于固定架上。
使用移液器轻轻吹打混匀细胞或细胞核的悬液，随后向芯片的细胞孔加入100μL细胞或细胞核的悬液，确保枪头接触孔底部。使用移液器轻轻吹打混匀磁珠，向芯片的磁珠孔加入100μL吹打混匀的磁珠，确保枪头接触孔底。立即添加350μL液滴生成油到芯片的油孔，迅速将注射器的推杆拉到卡槽位置，将推杆卡在卡槽处，启动计时器，5分钟后开始收集液滴，立即拧松收集管上的收集盖子，拔出芯片出口孔的连接管，竖直拉伸连接管，让管中的液滴流入收集管中，然后换上普通的收集管盖子。
如前所述，通过提高磁珠相的密度实现。根据泊松分布的计算公式，在此装置中液滴流速一致的情况下，当液滴大小为110μm时，颗粒的密度如果达到3000个/μL，有约70％的液滴会包裹到该颗粒。该实验所用磁珠密度为3000个/μL，经计算，液滴中包裹1个磁珠的概率是36.7％，包裹两个以上磁珠的概率是30％。当液滴包裹2个及以上磁珠的概率大于5％时，被认为实现了磁珠在液滴的超载。
液滴PCR
将液滴转移至PCR八连管(Axygen，PCR-0208-C)中，注意液滴液面不超过100μL，后在液滴表面加100μL矿物油(Sigma，M5904)覆盖，盖上八联管盖根据以下程序进行PCR：
95℃加热3分钟；
98℃加热30秒；
15个循环的以下步骤
{98℃加热10秒；
58℃退火30秒；
72℃延伸1.5分钟；}
72℃加热5分钟；
保持在4℃。
破乳
PCR结束后，将液滴转移到新的低吸附的1.5mL离心管(Invireogen，AM12400)中，加入50μL破乳试剂Perfluorooctanol(Sigma#370533)，用于溶解液滴，回收液滴中的磁珠。)，颠倒混匀，1000g，离心2分钟后放置在磁力架上，静置5分钟。保持磁珠吸附状态，弃去上清，加入1mL TE-TW，其是用TE缓冲剂(Invitrogen，AM9849)配制成终浓度为0.05％Tween-20(Sigma-Aldrich，9005-64-5)，颠倒混匀。以1000g的条件离心2分钟，之后放置在磁力架上，静置2分钟，弃去上清。重复上述步骤2次。
加入200μL酶切反应体系：20μL 10×EXO I缓冲液，10μL EXO I外切酶(Thermo Scientific，EN0581)，170μL水；在金属浴恒温器上(Eppendorf，5382000074)以37℃、1000rpm摇动的条件下孵育体系45分钟。反应结束后，短暂离心，加入1mL TE-SDS，其是用TE缓冲剂(Invitrogen，AM9849)配制成终浓度为0.5％SDS(Invitrogen，AM9820)，颠倒混匀，终止反应。
以1000g的条件离心2分钟，之后放置在磁力架上，静置2分钟，弃去上清。再次加入1mLTE-TW，颠倒混匀，以1000g的条件离心2分钟，之后放置在磁力架上，静置1分钟，弃去上清；重复上述步骤1次。
cDNA扩增
保持磁珠吸附状态，加入8份共计800μL的以下预配PCR体系：2X Kapa混合物(Roche，07958935001)50μL，0.4uM Tn引物4μL(序列为：5’-CGTAGCCATGTCGTTCTG-3’)，10％Opti-prep 18μL(Sigma，D1556-250ML)，和H 2O 28μL。吹打磁珠混匀，分装到八联管中，100μL/管；
根据以下程序进行PCR：
98℃加热30秒；
20个循环的以下步骤：
{98℃加热10秒，
58℃退火30秒，
72℃延伸1.5分钟；}
72℃加热5分钟；
保持在4℃。
反应终止后加入0.6X诺唯赞DNA纯化磁珠(Vazyme，#N411)纯化PCR产物，最后用50μL NF-H 2O洗脱cDNA。
cDNA文库构建
取100-200ng(约0.1-0.2pmo1)待片段化(打断)的cDNA，置于新的0.2mLPCR管中，体积应≤16μL，不足16μL部分用H 2O补足。按下表1在冰上配制片段化反应液，使用
Figure PCTCN2022076852-appb-000001
dsDNA片段化酶及其缓冲液(NEB，M0348S)：
表1.片段化反应液组分：
Figure PCTCN2022076852-appb-000002
将PCR管置于PCR仪上，热盖设为75℃，37℃孵育10分钟，获得片段化产物。反应结束后，向PCR管中加入30μL的0.1M EDTA，涡旋振荡混匀，终止反应。使用0.6x+0.2x的诺唯赞DNA纯化磁珠(Vazyme，#N411)纯化筛选经片段化的产物，并使用qubit dsDNA高敏检测试剂盒(Thermo，Q33230)进行浓度定量。
按如下表2在冰上配制末端修复反应液，使用Roche的ER&A加尾酶及其缓冲液(Roche，KK8500)：
表2.末端修复反应液组分：
Figure PCTCN2022076852-appb-000003
用移液器吸取10μL配制好的末端修复反应液加入片段化产物中，短暂涡旋混合均匀后，瞬时离心，之后将PCR管置于PCR仪上，热盖设为75℃，在37℃孵育30分钟，65℃加热15分钟，之后保持在4℃，获得末端修复产物。
按下表3在冰上配制接头连接反应液，使用NEB的连接缓冲液A(NEB，M0202S)和DNA连接酶(NEB，M0202S)：
表3.接头连接反应液组分：
Figure PCTCN2022076852-appb-000004
Figure PCTCN2022076852-appb-000005
用移液器缓慢吸取30μL配制好的接头连接反应液，加入经末端修复的产物中，涡旋震荡混匀，瞬时离心将反应液收集到管底，将PCR管置于PCR仪上，热盖设为75℃，23℃孵育30分钟，之后保持在4℃，获得接头连接产物。
使用1.0x的诺唯赞DNA纯化磁珠(Vazyme，#N411)纯化接头连接产物，并用qubitdsDNA高敏检测试剂盒(Thermo，Q33230)测定连接产物的浓度。
在离心管中按下表4配制接头引物PCR扩增反应混合液，进行接头引物PCR扩增：
表4.接头引物PCR扩增反应混合液组分：
Figure PCTCN2022076852-appb-000006
F引物：5’-PhoCGTAGCCATGTCGTTCTG*C*-3’，其中5’-端的PhoC是磷酸化修饰的C，3’-末尾两个碱基G*C*是硫代修饰的。
R引物：5’-TGTGAGCCAAGGAGTTGTTGTCTTCCTAAGACCGCT-3’。
用移液器吸取54μL配制好的接头引物PCR扩增反应混合液加入纯化后的接头连接产物中，涡旋震荡混匀，瞬时离心将反应液收集至管底。按以下方案进行接头引物PCR扩增：
95℃，3分钟；
10-15个循环的以下步骤：
{98℃加热20秒，
60℃退火20秒，
72℃延伸30秒；}
72℃加热5分钟；
保持在4℃。
接头引物PCR反应结束后，使用0.6x+0.6x诺唯赞DNA纯化磁珠(Vazyme，#N411)纯化产物，并用qubit dsDNA高敏检测试剂盒(Thermo，Q33230)对产物定量。
高通量测序
在测序前，需将所获得的cDNA产物变性。取200-400ng如上获得的cDNA产物，用NF-H 2O补充体积至47μL，加入3μL 20uM的夹板寡核苷酸(splint oligo)(5’-GACATGGCTACGTGTGAGCCAAGG-3’)，短暂涡旋混合均匀，瞬时离心5秒，之后，将PCR管置于PCR仪上，热盖设为105℃，在95℃下加热3分钟，之后迅速置于冰上5-10分钟。
按照如下表5配制单链环化反应体系，使用NEB的DNA连接酶和其连接缓冲液B(NEB，M0202L)：
表5.单链环化反应液组分：
Figure PCTCN2022076852-appb-000007
将配制好的单链环化反应液加到经变性的产物(即解链产物)中，短暂涡旋混合均匀，瞬时离心5秒，之后，将PCR管置于PCR仪上，热盖设为75℃，在37℃下孵育30分钟。
在单链环化反应快结束时，提前按如下表6在冰上配制酶切消化反应液，使用NEB的外切酶B及其缓冲液(NEB，M0293S)：
表6.酶切消化反应液组分：
Figure PCTCN2022076852-appb-000008
用移液器吸取4μL配制好的酶切消化反应液加入单链环化产物中，短暂涡旋混合均匀，瞬时离心5秒，之后，将PCR管置于PCR仪上，热盖设为75℃，在37℃下孵育30分钟。
酶切反应结束后，向PCR管中加入3μL终止溶液(0.1M EDTA)，涡旋混匀，瞬时离心5秒，收集液体至管底。
使用2.5x诺唯赞DNA纯化磁珠(Vazyme，#N411)对环化产物进行纯化，使用qubit dsDNA高敏检测试剂盒(Thermo，Q33230)对纯化后的产物进行定量，当文库质量＞0.5ng/μL时认为文库检测合格，可用于上机测序。
实施例2：使用人源293T细胞系的超高通量单细胞转录组测序
在本实施例中，采用人源293T细胞系进行了超高通量单细胞转录组测序。使用甲醇固定人源293T细胞系，在第一轮标记中使用了24种标签。细胞中的读取值为：0.769，每个细胞 的平均单分子标记数为：1008个，每个细胞的平均基因为：800个。最终单张芯片得到将近2万个可用细胞。
实施例3：使用人源293T和鼠源3T3混合细胞系的超高通量单细胞转录组测序
在本实施例中，采用人源293T和鼠源3T3混合细胞系进行了超高通量单细胞转录组测序。使用甲醇固定人源293T和鼠源3T3混合细胞系，在第一轮标记中使用了48种标签。细胞中的读取值为：0.626，每个细胞的平均单分子标记数为：1127个，每个细胞的平均基因为：469个。最终单张芯片得到约3万个可用细胞。人鼠细胞双包率为1.59％。
实施例4：使用人源293T和鼠源3T3混合细胞系的超高通量单细胞转录组测序
在本实施例中，采用人源293T和鼠源3T3混合细胞系进行了超高通量单细胞转录组测序。使用甲醇固定人源293T和鼠源3T3混合细胞系，在第一轮标记中使用了96种标签。细胞中的读取值为：0.766，每个细胞的平均单分子标记数为：661个，每个细胞的平均基因为：104个。最终单张芯片得到约8万个可用细胞。人鼠细胞双包率为10.65％。
从以上的描述中，可以看出本发明方法实现了不同细胞系以及组织来源的细胞的超高通量单细胞转录组测序，具体而言：
1)本发明方法实现了经原位逆转录后的细胞或细胞核与经预处理的磁珠的超载，提供了一种高效、灵活且易于使用的大规模scRNA测序方法。第一轮标记可以通过增加第一轮标记种类更进一步扩展通量。由于第一轮标记中每个孔都接收一个唯一的条形码，因此可以直接在单次实验中多重分析数百甚至数千个单独的样本——例如测量转录组对细胞系中大量遗传或药理学扰动的反应，或处理来自大型流行病学队列的组织样本。
2)本发明方法操作简单，效率高，实现了单次实验获得十万个细胞级别的超高通量。本方法在一个简单的两阶段工作流程中将组合索引的可扩展性与液滴发生器的效率和易用性相结合。本发明展示了单次实验相比普通droplet-seq实验通量增加了10倍。本发明所需实验设备简单，只需要标准实验室设备和微流体液滴发生器，只需两轮索引，即可在完整细胞或细胞核内完成逆转录。
以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种单细胞转录组测序文库的构建方法，其特征在于，所述构建方法包括以下步骤：
    1)对单细胞悬液进行细胞或细胞核内原位逆转录以产生带有第一标记的全长第一链cDNA；
    2)将步骤1)获得的经原位逆转录后的细胞或细胞核和用于第二链cDNA合成的PCR反应体系与能够识别所述第一标记的磁珠超载到液滴中，进行液滴PCR反应；
    3)在所述液滴PCR反应结束后回收所述磁珠；
    4)对在步骤3)中获得的所述磁珠上的cDNA进行二次扩增；和
    5)对在步骤4)中获得的经二次扩增后的cDNA进行片段化文库构建，得到所述单细胞转录组测序文库。
  2. 根据权利要求1所述的构建方法，其特征在于，所述步骤1)中，采用含标签的寡聚-dT引物进行所述原位逆转录从而产生带有所述第一标记的所述全长第一链cDNA；
    其中，所述寡聚-dT引物按照从5’到3’的顺序依次包括：
    a.磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；
    b.用于识别单个cDNA转录本的单分子标记；
    c.用于区分不同细胞或细胞核的第一轮标签；和
    d.用于捕获mRNA的多聚T序列；
    从而使得在所获得的具有所述第一标记的所述全长第一链cDNA的5’-端按照从5’到3’的顺序依次包括：磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；所述单分子标记；所述第一轮标签和所述多聚T序列，其中，所述磁珠能够识别所述第一标记中的所述磁珠捕获区。
  3. 根据权利要求1所述的构建方法，其特征在于，在步骤1)中，采用具有末端转移酶活性的逆转录酶进行所述原位逆转录，并且所述原位逆转录的体系中还包含模板置换引物(Tn-TSO)，
    优选地，所述具有末端转移酶活性的逆转录酶选自Maxima H酶、SSII酶或SSIV酶；
    优选地，所述模板置换引物经设计包含位于3’-端的鸟嘌呤，优选地，所述鸟嘌呤的个数为3个。
  4. 根据权利要求1所述的构建方法，其特征在于，所述原位逆转录的体系中还包含甘油，
    优选地，所述甘油在所述原位逆转录的体系中的终浓度为5％～10％；
    优选地，所述构建方法还包括在进行所述原位逆转录前对所述单细胞悬液中的细胞 或细胞核进行固定和/或透化预处理；
    更优选地，使用有机溶剂进行所述固定和/或透化预处理；
    进一步优选，所述有机溶剂为甲醇或多聚甲醛；
    更进一步优选地，所述甲醇的工作浓度为50％～100％，所述多聚甲醛的工作浓度为1％～4％。
  5. 根据权利要求1所述的构建方法，其特征在于，在步骤2)中，通过控制所述单细胞悬液中的细胞或细胞核的密度为100～1000个每μL，并且所述磁珠的密度为2000～5000个每μL；优选地，所述细胞或细胞核的密度为500个每μL，并且所述磁珠的密度为3000个每μL。
  6. 根据权利要求1所述的构建方法，其特征在于，步骤2)中的所述用于第二链cDNA合成的PCR反应体系包括：同时与所述全长第一链cDNA的3’-端以及磁珠引物互补的引物以及PCR试剂；
    优选地，所述同时与所述全长第一链cDNA的3’-端以及所述磁珠引物互补的引物为Tn引物。
  7. 根据权利要求1所述的构建方法，其特征在于，步骤2)中的所述磁珠为带有第二标记的磁珠，
    优选地，所述第二标记按照与磁珠距离由近及远的顺序依次包括：Tn引物、第二轮标签以及用于识别并捕获所述全长第一链cDNA的cDNA捕获序列；
    更优选地，所述第二轮标签包括第二轮第一标签和第二轮第二标签；
    进一步优选地，所述第二轮第一标签和所述第二轮第二标签之间还包括第一接头；
    更进一步优选地，所述Tn引物与所述磁珠之间还包括第二接头。
  8. 根据权利要求1-7中任一项所述的构建方法，其特征在于，步骤5)包括：
    将所述步骤4)中获得的经二次扩增后的cDNA进行片段化文库构建，得到链状cDNA文库，所述链状cDNA文库即为所述单细胞转录组测序文库；
    优选地，进一步将所述链状cDNA文库制备成环状cDNA文库，所述环状cDNA文库即为所述单细胞转录组测序文库；
    优选地，将所述链状cDNA文库制备成环状cDNA文库包括：
    将所述链状cDNA文库中的双链cDNA在环化辅助序列的作用下进行解链，得到解链产物；
    采用DNA连接酶对所述解链产物进行连接，得到单链环化产物；
    对所述单链环化产物进行酶切降解残留未环化的单链cDNA和双链cDNA，得到所述环化cDNA文库；
    更优选地，在对所述单链环化产物进行酶切降解之后，进一步对降解后的所述单链环化产物进行纯化，得到所述环化cDNA文库；
    进一步优选，采用磁珠对所述单链环化产物进行纯化，得到所述环化cDNA文库。
  9. 一种单细胞转录组文库的测序方法，其特征在于，所述测序方法包括：
    按照权利要求1至8中任一项所述的构建方法构建单细胞转录组测序文库；和
    将所述单细胞转录组测序文库进行上机测序。
  10. 一种制备单细胞转录组文库的试剂盒，其特征在于，所述试剂盒包括以下各项中至少一种：带有第一标记的含标签的寡聚-dT引物、模板转换引物、逆转录酶、甘油、固定和/或透化试剂、同时与全长第一链cDNA的3’-端以及磁珠引物互补的引物、带有第二标记的磁珠、链状cDNA文库构建试剂和环状cDNA文库构建试剂；
    优选地，所述含标签的寡聚-dT引物按照从5’到3’的顺序依次包括：
    a.磁珠捕获区，其是与磁珠上的第二标记中的寡核苷酸互补的互补区；
    b.用于识别单个cDNA转录本的单分子标记；
    c.用于区分不同细胞或细胞核的第一轮标签；和
    d.用于捕获mRNA的多聚T序列；
    优选地，所述逆转录酶为具有末端转移酶活性的逆转录酶，更优选所述具有末端转移酶活性的逆转录酶选自Maxima H酶、SSII酶或SSIV酶；
    优选地，所述模板转换引物经设计包含鸟嘌呤，优选地，所述鸟嘌呤的个数为3个；
    优选地，所述甘油在原位逆转录的体系中的终浓度为5％～10％；
    优选地，所述固定和/或透化溶剂为有机溶剂，更优选地，所述有机溶剂为甲醇或多聚甲醛，更进一步优选地，所述甲醇的工作浓度为50％～100％，所述多聚甲醛的工作浓度为1％～4％；
    优选地，所述同时与所述全长第一链cDNA的3’-端以及所述磁珠引物互补的引物为Tn引物；
    优选地，所述带有第二标记的磁珠中，所述第二标记按照与所述磁珠的距离由近及远的顺序依次包括：Tn引物、第二轮标签以及用于捕获所述第一链cDNA的cDNA捕获序列，更优选地，所述第二轮标签包括第二轮第一标签和第二轮第二标签，进一步优选地，所述第二轮第一标签和所述第二轮第二标签之间还包括第一接头，更进一步优选地，所述 Tn引物与所述磁珠之间还包括第二接头；
    优选地，所述链状cDNA文库构建试剂用于逆转录及扩增后的cDNA进行片段化文库构建，得到链状cDNA文库；
    优选地，所述环状cDNA文库构建试剂用于将所述链状cDNA文库制备成环状cDNA文库。
PCT/CN2022/076852 2022-02-18 2022-02-18 单细胞转录组测序文库的构建方法和测序方法，以及制备单细胞转录组文库的试剂盒 WO2023155135A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN111378556A (zh) * 2018-12-29 2020-07-07 青岛华大智造普惠科技有限公司 微流控芯片及其制备方法、单细胞微液滴的制备方法
US20200248175A1 (en) * 2017-10-23 2020-08-06 Massachusetts Institute Of Technology Calling genetic variation from single-cell transcriptomes
WO2021147069A1 (zh) * 2020-01-23 2021-07-29 青岛华大智造普惠科技有限公司 基于液滴微流控的单细胞测序及应用
WO2021189679A1 (zh) * 2020-03-27 2021-09-30 中国人民解放军陆军军医大学 一种单细胞转录组测序文库的构建方法及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200248175A1 (en) * 2017-10-23 2020-08-06 Massachusetts Institute Of Technology Calling genetic variation from single-cell transcriptomes
CN111378556A (zh) * 2018-12-29 2020-07-07 青岛华大智造普惠科技有限公司 微流控芯片及其制备方法、单细胞微液滴的制备方法
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
WO2021147069A1 (zh) * 2020-01-23 2021-07-29 青岛华大智造普惠科技有限公司 基于液滴微流控的单细胞测序及应用
WO2021189679A1 (zh) * 2020-03-27 2021-09-30 中国人民解放军陆军军医大学 一种单细胞转录组测序文库的构建方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CAO, JUNYUE ET AL.: "Comprehensive single cell transcriptional profiling of a multicellular organism", SCIENCE, vol. 357, 18 August 2017 (2017-08-18), XP055624798, DOI: 10.1126/science.aam8940 *
MACOSKO, E. Z. ET AL.: "Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets", CELL, vol. 161, 21 May 2015 (2015-05-21), XP055586617, DOI: 10.1016/j.cell.2015.05.002 *

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