Single-cell transcriptome sequencing and its applications

Abstract

The present invention provides a single-cell transcriptome sequencing method and its application, which includes the steps of preparing a sample of cells to be detected into a single-cell suspension, followed by fixing with a fixative, and synthesizing the first strand of cDNA by performing an in situ reverse transcription reaction on the RNA of the fixed single cells using a reverse transcription primer.

Description

[Technical field]

[0001] The present invention belongs to the field of biotechnology, and relates to the field of single cell sequencing, in particular to single cell transcriptome sequencing methods and applications thereof. [Background technology]

[0002] Currently, the most widely used high-throughput single-cell transcriptome sequencing technology is the single-cell sequencing technology based on the droplet microfluidic platform developed by 10X Genomics, which can mark, sequence and analyze thousands of cells to obtain gene expression profiles at the single-cell level, and realize the classification of cell subpopulations and the detection of differentially expressed genes between cell subpopulations. Similar technologies include inDrop technology and Drop-seq technology.

[0003] The basic operation and principle of 10X Genomics transcriptome sequencing technology (see Figure 1) is to prepare the sample into a single cell suspension (where the cell activity needs to be more than 90% and the cell concentration is generally about 700-1200 cells / μL), then a single coding microsphere with a barcode and a primer (the coding microsphere is composed of a part of a gel bead and a primer connected to the gel bead) and a single cell are encapsulated in an oil droplet through a microfluidic platform, in each droplet, the gel bead dissolves, the cell dissolves and the mRNA is released, and by reverse transcription, cDNA with barcode and UMI information for sequencing is generated, and after the droplet oil layer is crushed, the cDNA is collected and amplified to prepare a cDNA library, and then the library is sequenced and detected using an Illumina sequencing platform, thereby obtaining a large amount of single-cell gene expression data.

[0004] The primer sequence connected to the gel beads used in the above sequencing technology includes four parts: an Illumina TruSeq Read 1 sequencing primer, a 16-nt barcode, a 12-nt UMI, and a 30-nt Poly(dT) reverse transcription primer, where the TruSeq Read 1 sequencing primer is a known short peptide nucleotide sequence used for subsequent on-machine sequencing, the barcode corresponds one-to-one with the microbeads, there are a total of 4 million different barcodes, the UMI is a sequence composed of random bases, and each DNA molecule has its own unique UMI sequence, which is used to distinguish different samples during mixed sequencing (i.e., to distinguish which sequences obtained by sequencing are derived from the same original cDNA molecule), and the Poly(dT) reverse transcription primer is a homopolymer DNA fragment containing 30 T bases, which is used to capture transcripts with polyA tails. Summary of the Invention [Problem to be solved by the invention]

[0005] The reverse transcription primers used in high-throughput single-cell transcriptome sequencing technology, such as that of 10X Genomics, are all Poly(dT) reverse transcription primers, which can only obtain some 3'-end transcript information and cannot detect RNA that does not contain polyA (including damaged mRNA fragments and miRNAs and lncRNAs that do not contain polyA). Therefore, the sensitivity of the technology is very low in practical application, and it can usually detect less than 10% of mRNA. At the same time, the technology has high requirements for RNA quality, and if you want to get better sequencing results, the cell viability of the sequencing sample needs to be greater than 80%, and the sequencing effect is often poor when frozen samples or fixed samples are used. In addition, because bacterial RNA does not have polyA, traditional high-throughput single-cell transcriptome sequencing technology cannot be used for bacterial transcriptome sequencing. [Means for solving the problem]

[0006] In order to solve the above problems, in a first aspect, the present invention aims to provide a single-cell transcriptome sequencing method comprising the steps of preparing a sample of cells to be detected into a single-cell suspension, followed by fixing with a fixative, and synthesizing a first strand of cDNA by performing an in situ reverse transcription reaction on the RNA of the fixed single cells using a reverse transcription primer.

[0007] In certain embodiments, the reverse transcription primer of the present invention may be a reverse transcription random primer, a reverse transcription primer designed against a target RNA sequence, or a mixture of a reverse transcription random primer and a reverse transcription primer designed against a target RNA sequence.Preferably, the reverse transcription primer is a reverse transcription random primer.

[0008] In certain embodiments, the method of the present invention may comprise the step of adding a capture linker to the end of the first strand of the cDNA obtained by reverse transcription, said capture linker being complementary to the sequence of the capture linker complementary strand on the encoded microsphere, said capture linker being any fragment of a known sequence, preferably said capture linker being a Poly(dA) fragment, a Poly(dT) fragment, a Poly(dG) fragment or a Poly(dC) fragment.

[0009] In certain embodiments, the method of the present invention may include, after attachment of the capture linker, containing the single cell and a single of the encoded microspheres in a single chamber to form a single cell isolation, and synthesizing a second strand of cDNA in the single chamber.

[0010] In certain embodiments, the method of the invention may comprise the steps of synthesizing the second strand of said cDNA followed by PCR amplification, library construction and sequencing of the double stranded cDNA.

[0011] In certain embodiments, the single-stranded DNA on the encoded microsphere used in the methods of the present invention may comprise an upstream amplification primer complementary fragment, a barcode, a UMI and a capture linker complementary strand, preferably, the barcode may be one or more barcodes, more preferably, three barcodes.

[0012] In certain embodiments, the methods of the invention may achieve the isolation of said single cells using a microfluidic chip or microplate.

[0013] In certain embodiments, the fixative used in the method of the present invention may be a simple fixative or a mixed fixative, preferably, the simple fixative may include, but is not limited to, paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, and mercury, preferably, the mixed fixative may include, but is not limited to, acetic acid-alcohol mixture, formalin-acetic acid-alcohol solution, and Bouin's fixative.

[0014] In a particular embodiment, the method of the present invention includes the steps of preparing a sample of cells to be detected into a single cell suspension, followed by fixing with a fixative; synthesizing a first strand of cDNA by performing an in situ reverse transcription reaction on the RNA of the fixed single cell using a reverse transcription primer; adding a capture linker to the end of the first strand of cDNA obtained by reverse transcription, wherein the capture linker is complementary to the sequence of the capture linker complementary strand on the encoded microsphere; after adding the capture linker, including the single cell and the single encoded microsphere in a single chamber to form a single cell separation; synthesizing a second strand of cDNA in the single chamber to form double-stranded cDNA; and performing PCR amplification, library construction and sequencing on the obtained double-stranded cDNA.

[0015] In a second aspect, the present invention aims to provide an application of the method of the first aspect in whole transcriptome sequencing of single cells, single cell nuclei, single microorganisms, preferably in the fields of microbiology, basic medicine, clinical medicine, agriculture, cell biology, immunology, developmental biology, pathology, neurobiology, development, genetics, stem cells, oncology, reproductive health, macrogenomics, microecology and new drug research and development.

[0016] In a third aspect, the present invention aims to provide a diagnostic or therapeutic method comprising gene sequencing by the single-cell transcriptome sequencing method of the first aspect. Effect of the Invention

[0017] Compared with the prior art, the sequencing method of the present invention has the following advantages: (1) The method of the present invention can measure more types of cell samples, and can be applied to single cells and single cell nuclei of eukaryotic cells, prokaryotes (such as bacteria, actinomycetes, rickettsia, chlamydia, mycoplasma, cyanobacteria, and archaea), single cell algae, and pathogens. (2) The method of the present invention can be applied not only to fresh samples but also to fixed samples with low cellular activity, frozen samples, paraffin-embedded samples (FFPE), etc. (3) The method of the present invention can detect coding RNA and various forms of non-coding RNA, obtain complete transcriptome patterns, and perform quantification of transcriptome levels, analysis of gene differential expression, alternative splicing, gene fusions, RNA interactions, etc. (5) Compared with current microfluidic single-cell RNA sequencing technologies, the method of the present invention has the advantage of higher RNA detection sensitivity under the same sequencing depth. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing the principle of conventional 10X Genomics single-cell transcriptome sequencing. [Diagram 2] FIG. 1 is a schematic diagram showing the principle of the single-cell transcriptome sequencing method of the present invention. [Diagram 3] FIG. 2 is a schematic diagram showing the structure of an encoded microsphere for use in the present invention. [Figure 4] The results of processing and sequencing E. coli samples by adopting the method of the present invention: A. E. coli after lysozyme decomposition of cell wall under microscope; B. E. coli after reverse transcription under microscope; C. Amplification curve of qPCR experiment; D. Nucleic acid gel electrophoresis pattern; E. Gene detection number in E. coli. [Diagram 5] This is the result of sequencing a mixed sample of E. coli and B. subtilis using the method of the present invention. This is a scatter plot of the number of UMIs in single cells matched with the genomes of different bacterial species, where the number of UMIs is the number of cDNA molecules counted by sequencing. Each point in the figure represents one cell, where light-colored points represent cells that contain almost only B. subtilis cDNA, dark-colored points represent cells that contain almost only E. coli cDNA, and black points represent cells that are contaminated. [Figure 6] The results of sequencing a mixed sample of human mouse cell lines using the method of the present invention, in which: A. the number of detections of single-cell mouse genes obtained by sequencing a mixed sample of human mouse cell lines using the method of the present invention; B. the number of detections of single-cell mouse genes obtained by sequencing a mixed sample of human mouse cell lines using seven conventional sequencing methods such as 10X Genomics; C. the distribution status of the sequencing read lengths (Reads) of single-cell mouse genes in the sequencing results in different regions of the reference genome; D. the distribution uniformity statistical chart of the sequencing read lengths of single-cell mouse genes in the sequencing results on the reference gene 5'-3'. [Figure 7] The results of sequencing paraffin-embedded tissue (FFPE) samples of mouse liver tissue using the method of the present invention, in which A. Mouse liver tissue cell nuclei after dissociation under a microscope; B. Mouse liver tissue cell nuclei after reverse transcription reaction under a microscope; C. Amplification curve of qPCR experiment; D. Nucleic acid gel electrophoresis diagram; E. Gene detection number in mouse liver tissue cell nuclei. [Figure 8]1 shows the results of sequencing preparation of tobacco nuclei samples using the method of the present invention: A. Plant nuclei after dissociation under a microscope; B. Plant nuclei after reverse transcription under a microscope; C. Amplification curve of qPCR experiment; D. Nucleic acid gel electrophoresis. [Figure 9] The results of sequencing preparation of Chlamydomonas and Cyanobacteria samples by adopting the method of the present invention: A. Chlamydomonas and Cyanobacteria after cell wall digestion under a microscope; B. Chlamydomonas and Cyanobacteria after reverse transcription under a microscope; C. Amplification curves of qPCR experiments; D. Nucleic acid gel electrophoresis. [Figure 10] The results of sequencing preparation of cell samples fixed with different types of fixatives by adopting the method of the present invention: A. 3T3 cells after reverse transcription reaction under a microscope; B. Amplification curve of qPCR experiment; C. Nucleic acid gel electrophoresis. [Figure 11] 1 shows the results of sequencing preparation of cell samples in which different capture linker fragments were added to the ends of the cDNA strands using the method of the present invention, where A. C T values ​​of qPCR experiments; B. Nucleic acid gel electrophoresis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] An embodiment of the present invention provides a single-cell total RNA sequencing method, in which a reverse transcription primer is used to bind target RNA to a fixed single cell to generate an in situ reverse transcription reaction, then a capture linker is added to the end of the reverse-transcribed first strand cDNA, then a single chamber containing a single coding microsphere, a single cell and reaction reagents is made, and in the single chamber, the coding microsphere is bound to the first cDNA via the capture linker complementary strand and elongated to synthesize the second strand of cDNA with a barcode marker, and finally the resulting double-stranded cDNA is PCR amplified and sequenced in high throughput (as shown in FIG. 2). The sequencing method of the present application can be combined with microfluidic technology to build a high-throughput and highly sensitive single-cell transcriptome sequencing platform.

[0020] definition As used herein, single cells include, but are not limited to, single cells / single cell nuclei of eukaryotic cells, prokaryotes (such as bacteria, actinomycetes, rickettsia, chlamydia, mycoplasma, cyanobacteria and archaea), single cell algae, pathogens, and the like.

[0021] The cell sample in this specification includes, but is not limited to, fixed cell samples, cryopreserved samples, paraffin-embedded samples (FFPE), and the like.

[0022] RNA as used herein includes detectable coding RNA and various forms of non-coding RNA, such as miRNA, lncRNA, siRNA, circRNA, etc.

[0023] In this specification, the barcode fragment refers to a series of base sequences for distinguishing different cells, and as a cell label, this base sequence is required to be stable, synthesizable, and highly variable. Usually, you can design it yourself or select an appropriate barcode fragment from a barcode library. The barcode libraries of 10X Genomics and Xinqiuyuan, the two single-cell sequencing technologies currently mainstream on the market, can be found in their open source quantitative software Cellranger and CeleScope, respectively.

[0024] The reverse transcription random primer in this specification is constructed by connecting a PCR downstream amplification primer complementary fragment with six random base sequences (i.e., 5'-downstream amplification primer complementary fragment amplification primer fragment-NNNNNN-3', N=dG, dA, dT or dC), where the six random base sequences can bind to a target RNA sequence, and the downstream amplification primer complementary fragment is used to bind to the downstream amplification primer in a PCR amplification reaction.

[0025] Below, the main steps in the sequencing method of the present application are generally described. Preparation of coding microspheres The single-stranded DNA on the coded microspheres in the present invention consists of an upstream amplification primer complementary fragment, a barcode, a UMI (unique multiplex index) and a capture linker complementary strand (see FIG. 3). The upstream amplification primer complementary fragment binds to the upstream amplification primer in a PCR amplification reaction, the barcode is used to mark the cDNA in the same cell, the UMI is a random sequence to mark each of the original cDNAs, and the capture linker complementary strand binds to the capture linker connected to the first cDNA strand in the cDNA second strand synthesis reaction. The coded microspheres in the embodiment of the present invention can be adopted from microspheres in single cell sequencing reagent kits such as 10X Genomics, 1CellBio, Shin Kakugen, BD Rhapsody, etc.

[0026] Sample preparation Preparation of single cell suspension: In an embodiment of the present invention, in order to prepare a cell sample into a single cell suspension, corresponding digestive enzymes can be selected according to different types of cell samples. For example, for cultured cells, they can be digested into single cells with trypsin / EDTA; for fresh tissues, they can be digested with corresponding digestive enzymes (e.g., for muscle tissue, collagenase I and dispase are selected, and for liver tissue, collagenase IV is selected), then filtered and washed to prepare into single cells; for frozen storage samples, the samples must first be rapidly thawed in a water bath at 25-60°C; for paraffin-embedded samples (FFPE), they must first be dewaxed with xylene or other environmentally friendly dewaxing agents, and then decrosslinked; for single cell nuclear transcriptome sequencing, the single cell samples must first be treated with a strong non-ionic detergent (such as NP-40) to dissolve the cell membrane.

[0027] Fixation of single cells: In an embodiment of the present invention, a sample of cells to be detected is prepared into a single cell suspension and then fixed with a fixative. The sample is generally treated with a fixative to fix the macromolecular (RNA, protein, etc.) structures inside the cells / cell nuclei, so that the morphology, structure and composition of the intact single cells / cell nuclei can be maintained during subsequent experiments, and the RNA can be stably fixed in the cells / cell nuclei. During operation, based on the characteristics of different sample types, appropriate fixatives can be selected, including but not limited to simple fixatives such as paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, mercury, etc., and mixed fixatives such as acetic acid-alcohol mixture, formalin-acetic acid-alcohol solution, Bouin's fixative, and different fixation times such as 15min-30min or overnight.

[0028] Reverse transcription In an embodiment of the present invention, the reverse transcription primer is bound multiple times on the RNA of the immobilized cells to generate an in situ reverse transcription reaction, where the reverse transcription primer can be designed according to different actual requirements, for example, it can be a reverse transcription random primer, a reverse transcription random primer and a Poly(dT) primer added simultaneously, a target primer for a specific gene, etc. 10% TritonX-10 can also be added to the reverse transcription system to permeabilize the cell membrane of bacteria, so that the reaction reagent can easily enter the inside of the bacteria.

[0029] Addition of capture linkers Coding microspheres on single-stranded cDNA Capture Linker ComplementIn order to further synthesize the second strand of cDNA by binding with , it is necessary to add a capture linker to the 3' end of the first strand of cDNA after reverse transcription. In an embodiment of the present invention, the end transfer method can add Poly(dA), Poly(dT), Poly(dG) or Poly(dC) as a capture linker to the end of the first strand of cDNA, the DNA binding method can also add a specific capture linker to the end of the first strand of cDNA, and the template displacement method can also be adopted to use reverse transcriptase in the reverse transcription step to add three dCs as capture linkers to the end of the first strand of cDNA after reverse transcription. In contrast, on the single-stranded cDNA of the coding microspheres The capture linker complementary strand is It can be adjusted accordingly.

[0030] Single cell isolation The separation of single cells in this specification generally refers to forming a single chamber containing a single coding microsphere, a single cell, and reaction reagents. To complete the subsequent extension reaction to synthesize the second strand of cDNA, the extension reaction reagents in the single chamber usually include DNA polymerase, dNTPs, and reaction buffer. In the method of the present invention, according to the size and type of different cells, different microfluidic chips can be designed to generate microdroplets to separate single cells, or microplate (microwell) technology can be used to separate single cells. Generally, according to the number of cells to be detected for sequencing, a corresponding number of single droplets are collected, or a corresponding number of pores are prepared.

[0031] Synthesis of second strand of cDNA The capture linker complement on the single-stranded cDNA to which the coding microsphere is attached binds to the capture linker added to the first strand of cDNA, which is then extended under the action of DNA polymerase to synthesize a second strand of cDNA. The resulting second strand of cDNA contains the upstream amplification primer complement fragment, the barcode, the UMI, the capture linker complement and the cDNA sequence.

[0032] Library construction and high-throughput sequencing First, the original double-stranded cDNA after the previous extension reaction is purified by magnetic beads method, and the upstream primer, downstream primer and PCR amplification reaction reagents are added to PCR amplify the original double-stranded cDNA. The PCR amplification product is purified by magnetic beads method, and the TA cloning connection linker library construction method is adopted to repair the ends of the PCR amplification product and add A tails to connect the linker (adapter). The constructed library can be sequenced by high-throughput using Illumina sequencing platform or HuaDa ZhiZheng sequencing platform. The upstream primer and downstream primer of the PCR amplification reaction are designed and synthesized based on the upstream amplification primer complementary fragment on the single-stranded cDNA of the coding microsphere and the downstream amplification primer complementary fragment in the reverse transcription random primer, respectively.

[0033] Specific embodiments In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

[0034] Based on the embodiments of the present invention, any other embodiments that a person skilled in the art can think of without any creative effort are within the scope of protection of the present invention.

[0035] The technical solutions of the present invention are further described below through specific embodiments in conjunction with the accompanying drawings.

[0036] Example 1: Sequencing of E. coli samples Sample preparation Approximately 1 million different strains (1, 2, 3, 4) of E. coli were collected respectively to verify that the present invention patent can be used for single-cell transcriptome sequencing of bacterial samples, and PBS buffer (PBST) containing 0.05% Tween-20 (purchased from Shanghai Biotechnology Co., Ltd.) was added to wash three times, and aggregated bacteria were removed by shaking and filtration, and the bacteria in the sample were made into a single bacterial suspension, and 1% paraformaldehyde (purchased from Beijing Solaibao Technology Co., Ltd.) was added and fixed overnight at 4 ° C. The bacterial sample after fixation was washed three times with PBST buffer, and lysozyme (purchased from Thermo Fisher Scientific, USA) was added to dissolve the cell wall, and the sample was treated at 37 ° C for 15 minutes, and PBST buffer was added to wash three times.

[0037] Reverse transcription After pretreatment, the reverse transcription-permeabilization reaction reagent containing reverse transcriptase (purchased from Thermo Fisher Scientific, USA), reverse transcription reaction buffer, dNTPs (purchased from Beijing Solaibao Technology Co., Ltd), reverse transcription random primer (synthesized by Shanghai Biotechnology Co., Ltd.), and 10% TritonX-10 (purchased from Shanghai Biotechnology Co., Ltd.) was added to the sample to perform the reverse transcription reaction, and 50 ul of reverse transcription reaction system was used for 500,000 single bacteria. After the reaction was completed, PBST buffer was added to wash three times.

[0038] Addition of capture linkers Terminal transferase (purchased from Thermo Fisher Scientific, USA), dCTP (purchased from Shanghai Biotechnology Co., Ltd.) and reaction buffer were added to the sample after reverse transcription, and the mixture was incubated at 37°C for 30 minutes to add a Poly(dC) capture linker fragment to the 3' end of the first cDNA strand. After the reaction was completed, the mixture was washed three times with PBST buffer. Microscopic examination showed that the E. coli sample maintained a dispersed and intact monobacterial morphology after cell wall lysis and reverse transcription (see Figure 4A).

[0039] Single cell isolation Count each sample and adjust the bacterial concentration to 200-400 / ul. Bacterial sample, extension reaction reagent (including DNA polymerase (purchased from Thermo Fisher Scientific, USA), dNTPs and reaction buffer), coding microspheres (purchased from 1CellBio, USA), and oil phase (electron fluoride solution 7500 containing 0.2% surfactant, purchased from 3M, USA) are added to syringes, which are connected to the corresponding inlets of the microfluidic chip via hoses. Set the appropriate flow rate to form a single droplet of water-in-oil containing a single bacterium, a single coding microsphere, and the extension reaction reagent, and collect the single droplet of about 200 ul.

[0040] Synthesis of second strand of cDNA To synthesize the second strand of the cDNA with a barcode marker in the single droplet, the collected single droplets are dispensed into different tubes and then elongated. After the elongation reaction is completed, 20% PFO (electron fluorination solution 7500 containing 20% ​​1H,1H,2H,2H-Perfluorooctanol, purchased from Sigma-Aldrich, USA) is added to each tube to break up the single droplets, and the cDNA in the tubes is purified and extracted by the magnetic bead method (magnetic beads are purchased from Beckman Coulter, USA).

[0041] Library construction and high-throughput sequencing A portion of the double-stranded cDNA (200-400 cells) was taken as a template, and a qPCR experiment was performed to detect the total captured cDNA content. The results show that the obtained CT value is about 17 (see FIG. 4B). Based on the obtained CT value, the appropriate number of amplification cycles (generally, the CT value plus 3 cycles is taken) is calculated, and the remaining cDNA samples in the other tubes are further amplified by PCR amplification reaction to amplify the encoded cDNA to an appropriate total content (about 100 ng). The results of nucleic acid electrophoresis performed by taking 3 ul of the amplified product show that a dispersed ribbon was obtained, and the size distribution of the ribbon was mainly 200-500 bp (see FIG. 4C), which indicates that the cDNA captured from the E. coli sample by the method of the present invention has a high capture rate and a wide coverage, and can be used for subsequent high-throughput sequencing.

[0042] 100ng of cDNA after amplification is taken, and the TA cloning-connected linker library construction method is adopted to repair the ends and add A tails to connect the linkers (library construction reagent kit is purchased from Illumina, USA). The constructed library is subjected to high-throughput sequencing using the Illumina sequencing platform. The sequencing results show that in a single E. coli, the number of genes can be detected to be distributed between 1000 and 3000 (see Figure 4E), basically including all genes expressed in E. coli, which indicates that the method of the present invention is applicable to bacterial single-cell transcriptome sequencing and has high sensitivity.

[0043] Example 2: Sequencing of a mixed sample of E. coli and B. subtilis Approximately 1 million mixed samples of E. coli and Bacillus subtilis are collected to verify the accuracy of single-cell transcriptome sequencing using the present invention patent. Sample pretreatment, reverse transcription, addition of capture linker, single cell isolation, second strand cDNA synthesis, library construction and high-throughput sequencing are performed using the method described in Example 1 above. The sequencing results show that after genome matching, a total of 251 bacteria were detected from the mixed sample of E. coli and Bacillus subtilis, of which 4 bacteria contained both E. coli cDNA and Bacillus subtilis cDNA, which is usually due to the two bacteria entering the same droplet, and the remaining bacteria mostly only contained E. coli cDNA or Bacillus subtilis cDNA, and the overall contamination rate is probably less than 2% (see Figure 5). This shows that single-cell transcriptome sequencing using the present invention patent has high accuracy, can well separate E. coli and Bacillus subtilis mixed in the sample, and has little cross-contamination between species.

[0044] Example 3: Sequencing of a mixed sample of human-mouse cell lines Approximately 1 million human mouse cell line mixed samples (50% human HEK293 cells and 50% mouse 3T3 cells) frozen at -80°C are collected, the frozen cell samples are taken out and quickly thawed in a 37°C water bath, washed three times with PBS buffer, and fixed overnight at 4°C with 1% paraformaldehyde. The frozen cell samples after fixation are washed three times with PBST buffer. Then, reverse transcription, capture linker addition, single cell isolation, cDNA second strand synthesis, library construction and high-throughput sequencing are performed according to the method described in Example 1 above. After genome matching, the results of separating the data of mouse 3T3 cells showed that the method of the present invention can detect the number of mouse single-cell genes distributed between 4000 and 10000 (see Figure 6A) in cryopreserved cell samples. The literature Systematic comparison of single-cell and single-nucleus RNA-sequencing methods (Jiarui Ding et al., Nat Biotechnol. 2020 June; 38(6): 737-746.) showed that the number of single-cell transcriptome sequencing methods in the seven conventional technologies (the seven sequencing methods are two microplate-based low-throughput methods (Smart-seq2 and CEL-Seq2) and five high-throughput methods (10x Using a set of 3D sequencing methods (including Chromium, Drop-seq, Seq-Well, inDrops and sci-RNA-seq), we sequenced a mixed sample of human and mouse cell lines (50% human HEK293 cells and 50% mouse 3T3 cells), and compared the sequencing results of mouse single cells detected by the method of the present invention with the sequencing data of mouse single cells detected by the above seven methods. We reported that the sensitivity of the sequencing method of the present invention reached the sensitivity of low-throughput single-cell sequencing methods such as Smart-seq2 under the same sequencing depth, and was much higher than the sensitivity of high-throughput single-cell sequencing methods such as 10X Genomics (10X Chromium) (see Figure 6B).The sequencing matching results show that the sequencing read lengths of mouse single cells are distributed in different regions of the reference genome, including coding RNA, intergenic regions, interintronic and untranslated regions (see Figure 6C), indicating that the present invention patent can be adopted to sequence the whole transcriptome, and the sequencing matching results show that the sequencing read lengths of mouse single cells are evenly distributed on the 5'-3' of the reference genes (see Figure 6D).

[0045] Example 4: Sequencing preparation of paraffin-embedded tissue (FFPE) samples of mouse liver tissue About 20 mg of paraffin-embedded mouse liver tissue samples are collected. First, the paraffin-embedded samples are dewaxed and rehydrated, and the tissue is dissolved into single cell nuclei by adding cell lysis solution, and then washed three times by adding PBST. Then, reverse transcription, capture linker addition, single cell isolation, second strand cDNA synthesis, library construction and high-throughput sequencing are performed with reference to the method described in Example 1 above. The results show that the single dispersed cell nuclei successfully isolated from the FFPE sample can maintain the dispersed complete single cell nucleus morphology even after reverse transcription (see Figures 7A and 7B). After obtaining the purified double-stranded cDNA product, a qPCR experiment is performed using a portion of the cDNA (about 100 cell nuclei) as a template to detect the total cDNA content captured, and the obtained CT value is about 10 (Figure 7C), which indicates that the method of the present invention has a high capture rate for cDNA in the single cell nuclei of the FFPE sample. The cDNA product was PCR amplified (15 cycles) according to the CT value, 3ul of the amplified product was collected, and nucleic acid electrophoresis was performed to obtain a dispersed ribbon, and the size distribution of the ribbon was mainly 200-500bp (Figure 7D), which indicates that the method of the present invention has a high capture rate and wide coverage of cDNA captured from single cell nuclei of FFPE samples, and can be used for subsequent sequencing. The sequencing results show that the method of the present invention can detect 2000-3000 genes (see Figure 7E) in single cell nuclei of FFPE samples, which indicates that the method of the present invention can be applied to single cell nucleus transcriptome sequencing of FFPE samples.

[0046] Example 5: Sequencing preparation of tobacco cell nuclei samples Fresh tobacco samples are dissociated into single cell nuclei (see FIG. 8A) using a single cell preparation device, and a sample of about 1 million tobacco cell nuclei is collected, washed three times with PBS buffer, and fixed overnight at 4°C with 1% paraformaldehyde. After fixation, the tobacco cell nuclei sample is washed three times with PBST buffer. Then, reverse transcription, addition of capture linker, isolation of single cells, synthesis of second strand of cDNA, and construction of library are performed with reference to the method described in Example 1 above. The results show that the tobacco cell nuclei sample can maintain a dispersed and complete single cell nuclei morphology even after reverse transcription reaction (see FIG. 8B). The results of the qPCR experiment show that the CT value of the obtained tobacco cell nuclei is about 16 (see FIG. 8C), which indicates that the method of the present invention can capture a large amount of cDNA in the tobacco cell nuclei sample. The results of nucleic acid gel electrophoresis obtained dispersed ribbons with a size distribution mainly of 150-300 bp (Figure 8D), indicating that the method of the present invention has a high capture rate and wide coverage of cDNA captured from single cells of tobacco cell nuclei samples, which can be used for subsequent sequencing.

[0047] Example 6: Preparation of a mixed sample of Chlamydomonas and Cyanobacteria for sequencing Approximately 1 million Chlamydomonas and Cyanobacteria samples The invention patent was Chlamydomonas and Cyanobacteria samplesThe method is verified to be usable for single-cell transcriptome sequencing of Chlamydomonas and cyanobacteria. The Chlamydomonas and cyanobacteria samples are washed three times with PBST buffer, and then fixed with 1% paraformaldehyde at 4°C overnight. After fixation, the Chlamydomonas and cyanobacteria samples are washed three times with PBST buffer. The Chlamydomonas sample is added with a cell wall enzyme digestion solution containing 4% cellulase, 0.5% pectinase, and 0.3 mol / L mannitol at pH=6.5, treated at 37°C for 15 minutes, and washed three times with PBST buffer. The cyanobacteria sample is added with lysozyme to dissolve the cell wall, treated at 37°C for 15 minutes, and washed three times with PBST buffer. After that, the Chlamydomonas and cyanobacteria samples with dissolved cell walls are subjected to reverse transcription, addition of capture linkers, single cell separation, synthesis of the second strand of cDNA, and library construction, referring to the method described in Example 1 above. The results show that the Chlamydomonas and Cyanobacteria samples can maintain a dispersed and intact single-cell morphology (see Figures 9A and B) even after cell wall lysis and reverse transcription. The results of the qPCR experiment show that the obtained CT value of the Chlamydomonas sample is about 17, and the CT value of the Cyanobacteria sample is about 17 (Figure 9C), which indicates that the method of the present invention has a high capture rate for cDNA in the Chlamydomonas and Cyanobacteria samples. The results of the nucleic acid gel electrophoresis obtained dispersed ribbons with a size distribution mainly of 200-500 bp (Figure 9D), which indicates that the method of the present invention has a high capture rate and wide coverage of cDNA captured from single cells of the Chlamydomonas and Cyanobacteria samples, and can be used for subsequent sequencing.

[0048] Example 7: Sequencing preparation of mouse 3T3 cell line samples (employing different fixatives and different capture linkers) Four sets of cultured mouse 3T3 cell lines are taken, and the effect of using different fixatives to treat the samples is verified. Trypsin is added to the cell culture dish and digested at 37°C for 3 minutes, which is digested into single cells, and then PBS buffer is added and washed three times, and 1% paraformaldehyde, 70% ethanol, acetone, and 1% acetic acid (sample numbers 1, 2, 3, and 4) are added, respectively, and fixed at 4°C overnight. After the fixation is completed, PBST3 is added and washed three times. Then, reverse transcription, capture linker addition, single cell isolation, cDNA second strand synthesis, and library construction are performed by referring to the method described in Example 1 above. The results show that the cell samples fixed with different types of fixatives can maintain the complete single cell morphology (see Figure 10A) after the reverse transcription reaction. The method of the present invention has a high capture rate for cDNA from cell samples fixed with different types of fixatives (see Figure 10B) and obtained dispersed ribbons with a size distribution mainly between 200 and 500 bp (see Figure 10C), indicating that the method of the present invention has a high capture rate and wide coverage for cDNA captured from cell samples fixed with different types of fixatives, and can be used for subsequent high-throughput sequencing.

[0049] In addition, seven sets of cultured mouse 3T3 cell lines are collected to verify the effect of adding different capture linkers to the end of the first strand of cDNA. Trypsin is added to the cell culture dish and digested at 37°C for 3 minutes, which is digested into single cells, washed three times with PBS buffer, added 1% paraformaldehyde, fixed overnight at 4°C, and after fixation, washed twice with PBST3. Six sets of fixed cell samples are collected and reverse transcription reaction is carried out according to the method described in Example 2 above. Four sets of cell samples after reverse transcription are collected, and dATPs, dTTPs, dGTPs, dCTPs, terminal transferase and reaction buffer are added to each set, incubated at 37°C for 30 minutes, and Poly(dA), Poly(dT), Poly(dG) and Poly(dC) are added to the cDNA ends as capture linker fragments, respectively. After the reverse transcription is completed, another two sets of cell samples are collected, and DNA ligase, a specific capture linker fragment, and a reaction buffer are added, and the samples are incubated at 37°C for 30 minutes to add a specific capture linker fragment to the end of the cDNA. After the fixation is completed, another set of cell samples is collected, and a reverse transcription-permeabilization reaction reagent containing reverse transcriptase (Moloney Murine Leukemia Virus,), a reverse transcription random primer, dNTPs, a reverse transcription reaction buffer, and 10% TritonX-10 is added to perform a reverse transcription reaction, and when the reverse transcription is completed, three dCs are added as capture linker fragments to the end of the cDNA. The cell samples to which the capture linker has been added are subjected to single cell separation, cDNA second strand synthesis, and library construction according to the methods described in Examples 4, 5, and 6 above. The results showed that the CT values ​​of the cell samples with different capture linker fragments ranged from 12 to 13, indicating that the method of the present invention could capture a large amount of cDNA (see Figure 11A) from cells using different capture linker fragments, and the gel electrophoresis results showed that the size distribution of the dispersed ribbons was mainly 200 to 500 bp (see Figure 11B), indicating that the method of the present invention had a high capture rate and wide coverage of cDNA captured from cells using different capture linker fragments, and all of them could be used for subsequent high-throughput sequencing.

[0050] The above shows the basic principle and main features of the present invention, as well as the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above examples, and the above examples and the description of the specification are merely illustrative of the principles of the present invention, and that the present invention may have various modifications and improvements without departing from the spirit and scope of the present invention, and all of these modifications and improvements are included in the scope of the protection claims of the present invention. The scope of the protection claims of the present invention is defined by the appended claims and their equivalents.

Claims

1. preparing a sample of cells to be detected into a single cell suspension and then fixing the sample in a fixative; and synthesizing a first strand of cDNA by performing an in situ reverse transcription reaction on the RNA of the fixed single cell using a reverse transcription primer.

2. The method of claim 1, characterized in that the reverse transcription primer is a reverse transcription random primer, a reverse transcription primer designed against a target RNA sequence, or a mixture of a reverse transcription random primer and a reverse transcription primer designed against a target RNA sequence.

3. The method according to claim 1 or 2, further comprising the step of adding a capture linker to the end of the first strand of the cDNA obtained by reverse transcription, the capture linker having a complementary sequence to the capture linker complementary strand on the encoded microsphere, the capture linker being any fragment of a known sequence, preferably the capture linker being a Poly(dA) fragment, a Poly(dT) fragment, a Poly(dG) fragment or a Poly(dC) fragment.

4. 4. The method of claim 3, further comprising the step of, after adding the capture linker, containing the single cell and a single encoded microsphere in a single chamber to form a single cell isolation, and synthesizing a second strand of cDNA in the single chamber.

5. 5. The method of claim 4, further comprising the steps of: synthesizing the second strand of the cDNA; and then performing PCR amplification, library construction and sequencing on the double-stranded cDNA.

6. The method according to any one of claims 3 to 5, characterized in that the single-stranded DNA on the encoded microspheres used comprises an upstream amplification primer complementary fragment, a barcode, a UMI and a capture linker complementary strand, preferably said barcode being one or more barcodes, more preferably three barcodes.

7. The method according to any one of claims 3 to 6, characterized in that the isolation of said single cells is achieved using a microfluidic chip or a microplate.

8. The method according to any one of claims 1 to 7, characterized in that the fixative is a simple fixative or a mixed fixative, preferably the simple fixative includes but is not limited to paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate and mercury, preferably the mixed fixative includes but is not limited to acetic acid-alcohol mixture, formalin-acetic acid-alcohol solution and Bouin's fixative.

9. Application of the method according to any one of claims 1 to 8 for whole transcriptome sequencing of single cells, single cell nuclei, single microorganisms, preferably in the fields of microbiology, basic medicine, clinical medicine, agriculture, cell biology, immunology, developmental biology, pathology, neurobiology, development, genetics, stem cells, oncology, reproductive health, macrogenomics, microecology and drug development.

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