CN114774527A - High-throughput single-cell transcriptome sequencing method and application thereof - Google Patents

Abstract

The invention provides a high-throughput single-cell transcriptome sequencing method and application thereof, wherein the method comprises the steps of preparing a cell sample to be detected into a single-cell suspension and fixing the single-cell suspension by using a fixing solution; and using reverse transcription primer to make in-situ reverse transcription reaction on the fixed single-cell RNA to synthesize cDNA first chain; wherein the reverse transcription primer comprises a tag sequence.

Description

High-throughput single-cell transcriptome sequencing method and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to the field of single cell sequencing, and particularly relates to a single cell transcriptome sequencing method of multiple types of cells and application thereof.

Background

The single cell sequencing technology based on a droplet microfluidic platform, which is developed by 10 Xgenomics, can realize the marking, sequencing and analysis of thousands of cells, obtain a gene expression profile at the single cell level, realize the division of cell subsets and the detection of differentially expressed genes among the cell subsets, and the technologies similar to the technology comprise an inDrop technology and a Drop-seq technology.

The basic operation and principle of the 10X Genomics transcriptome sequencing technology is (see fig. 1): preparing a sample into a single-cell suspension (the cell activity of the sample is higher than 90%, and the cell concentration is generally about 700-1200 cells/mu L), and then wrapping a single coding microsphere (the coding microsphere is formed by a section of primer connected on gel beads and the gel beads) with a bar code (Barcode) and a primer and a single cell in an oil drop by a microfluidic platform; in each individual droplet, the gel beads are lysed and the cells lysed to release the mRNA, by reverse transcription, to produce cDNA with barcoded and UMI information for sequencing; and (3) crushing the oil drop layer, collecting cDNA (complementary deoxyribonucleic acid) for amplification, preparing a cDNA library, and then carrying out sequencing detection on the library by using an Illumina sequencing platform so as to obtain a large amount of single-cell gene expression data.

The primer sequences attached to the gel beads used in the above sequencing techniques comprise four parts: the kit comprises Illumina TruSeq Read 1 sequencing primers, a bar code of 16nt, UMI of 12nt and Poly (dT) reverse transcription primers of 30nt, wherein the TruSeq Read 1 sequencing primers are a section of known short peptide nucleotide sequence and are used for subsequent on-computer sequencing; the bar codes correspond to the microbeads one by one, and the total number of the bar codes is 400 tens of thousands; UMI is a sequence composed of random bases, each DNA molecule on the gel bead has its own UMI sequence, and the function of the UMI sequence is to distinguish different transcripts during mixed sequencing (namely to distinguish which sequenced sequences are from the same original cDNA molecule); poly (dT) reverse transcription primers are homopolymeric DNA fragments containing 30T bases and are used to capture transcripts with polyA tails.

The reverse transcription primers used in the high-throughput single-cell transcriptome sequencing technology represented by 10 × Genomics are all poly (dt) reverse transcription primers, so that only part of 3' terminal transcript information can be obtained, and RNA not containing polyA (including damaged mRNA fragments and miRNA, incrna, etc. not containing polyA) cannot be detected, which results in that the sensitivity of the technology in practical application is very low, and usually less than 10% of mRNA can be detected. Meanwhile, the technology has high requirements on RNA quality, if a better sequencing result is required, the cell viability of a sequencing sample is more than 80%, and the sequencing effect is often poor when a frozen or fixed sample is adopted. In addition, since the RNA of bacteria does not carry polyA, the existing high-throughput single-cell transcriptome sequencing technology cannot be used for sequencing the transcriptome of bacteria. At the same time, in order to avoid contamination in this technique, it is necessary to include as much as possible only one cell per droplet when performing cell separation, for which reason the droplet size is much higher than the cell size during preparation and typically only one type of single cell is sequenced at a time, which results in only about 1/10 of droplets being eventually populated with cells, and a large number of microspheres and reagents in empty droplets being wasted.

Therefore, there is a need in the art for a single-cell transcriptome sequencing method with higher capture efficiency, lower cost and contamination rate, and higher throughput of cell samples.

Disclosure of Invention

In order to solve the above problems, in a first aspect, the present invention provides a single cell transcriptome sequencing method, comprising preparing a cell sample to be tested into a single cell suspension, and fixing with a fixing solution; and using a reverse transcription primer to carry out in-situ reverse transcription reaction on the fixed RNA of the single cell to synthesize a first cDNA chain; wherein the reverse transcription primer comprises a tag sequence.

In particular embodiments, the reverse transcription primer in the methods of the invention may comprise a tag sequence and an RNA binding sequence, which may be a random RNA binding sequence, an RNA binding sequence designed for a target RNA sequence, or a combination thereof.

In a specific embodiment, the method of the present invention may further comprise adding a capture linker complementary to the sequence encoding the complementary strand of the capture linker on the microsphere to the tail end of the first strand of the cDNA of the single cell obtained by reverse transcription; the capture adaptor is any fragment of known sequence, preferably the capture adaptor is a Poly (dA) fragment, a Poly (dT) fragment, a Poly (dG) fragment or a Poly (dC) fragment.

In particular embodiments, the methods of the invention may further comprise containing a single cell and a single said encoded microsphere in a single chamber to form a cell compartment; and synthesizing a second strand of cDNA in the single chamber after forming the cell partition; wherein two or more single cells are included in at least a portion of the single chamber after forming the cell partition.

In particular embodiments, the two or more single cells in the methods of the invention may be the same type or different types of cells.

In particular embodiments, the methods of the invention may further comprise PCR amplification and pooling and sequencing of the double stranded cDNA after synthesis of the second strand of the cDNA.

In particular embodiments, the single-stranded DNA on the encoded microspheres used in the methods of the invention may comprise the upstream amplification primer complement, the barcode, the UMI, and the capture adaptor complement. In a preferred embodiment, the barcode may be one or more barcodes; in a more preferred embodiment, the barcode may be three barcodes.

In particular embodiments, microfluidic chips or microplates may be used in the methods of the invention to accomplish the cell separation.

In particular embodiments, the fixative used in the methods of the invention may be a simple fixative or a mixed fixative; preferably the simple fixative solution may include, but is not limited to, paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate and mercuric chloride; preferably, the mixed fixative may include, but is not limited to, acetic acid-alcohol mixture, formalin-acetic acid-alcohol solution, and Boin's fixative.

In particular embodiments, the methods of the invention may comprise: preparing a cell sample to be detected into a single cell suspension, and fixing the single cell suspension by using a fixing solution; synthesizing a first cDNA strand by performing in-situ reverse transcription reaction on the immobilized single-cell RNA by using a reverse transcription primer, wherein the reverse transcription primer sequentially comprises a downstream amplification primer complementary segment, a tag sequence and an RNA binding sequence from the 5 'end to the 3' end; adding a capture linker to the tail end of the first strand of the cDNA obtained by reverse transcription, wherein the capture linker is complementary to the sequence of the complementary strand of the capture linker on the coding microspheres; containing the single cell and a single said encoded microsphere in a single chamber to form a cell partition, wherein at least a portion of the single chamber contains two or more single cells after the formation of the cell partition; synthesizing a second strand of cDNA in the single chamber to form a double-stranded cDNA; and carrying out PCR amplification, library construction and sequencing on the obtained double-stranded cDNA.

In a second aspect, the present invention aims to provide the use 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 and development, genetics, stem cells, tumors, reproductive health, metagenomics and microecology, and the development of new drugs.

Compared with the prior art, the sequencing method has the following advantages:

(1) the cell sample measurable by the method has wider types, and can be applied to unicells and mononuclear cells of eukaryotic cells, prokaryotes (bacteria, actinomycetes, rickettsia, chlamydia, mycoplasma, cyanobacteria, archaea and the like), unicellular algae, viruses and the like; the method is not only suitable for fresh samples, but also suitable for fixed samples with low cell activity, frozen preserved samples, paraffin embedded samples (FFPE) and the like;

(2) the method of the invention can detect coding RNA and non-coding RNA in various forms and obtain more complete transcriptome map, and carry out analysis such as transcriptome level quantification, gene differential expression, alternative splicing, gene fusion, RNA interaction and the like;

(3) compared with the current microfluidic single-cell RNA sequencing technology, the method has higher RNA detection sensitivity under the same sequencing depth;

(4) the method has higher cell sample flux which can be detected once, can simultaneously sequence the single-cell transcriptome of various cells, and effectively reduces the detection time and the economic cost while ensuring the sequencing accuracy and the sensitivity.

Drawings

FIG. 1 is a schematic diagram of the conventional 10 Xgenomics single cell transcriptome sequencing method.

FIG. 2 is a schematic diagram of the principle of the single cell transcriptome sequencing method of the present invention.

FIG. 3 is a schematic diagram of the structure of an encoded microsphere used in the present invention.

FIG. 4 shows the results of sequencing and treating E.coli samples by the method of the present invention. A. Escherichia coli with cell wall degraded by lysozyme under microscope; B. carrying out reverse transcription on the reacted escherichia coli under a microscope; amplification curve of qpcr experiment; D. a nucleic acid gel electrophoresis image; E. number of gene detected in E.coli.

FIG. 5 shows the results of sequencing a mixed sample of Escherichia coli and Bacillus subtilis by the method of the present invention; comparing the single cells to UMI number scatter diagrams on genomes of different strains, wherein the UMI number is the number of cDNA molecules counted by sequencing, each point in the diagrams represents a cell, the light-colored points represent the cells which almost only contain cDNA of bacillus subtilis, the dark-colored points represent the cells which almost only contain cDNA of escherichia coli, and the black points represent the contaminated cells.

FIG. 6 shows the results of sequencing a mixed sample of human and murine cell lines using the method of the present invention; a, sequencing a human-mouse cell line mixed sample by adopting the method to obtain the detected number of mouse single cell genes; B. adopting seven sequencing methods such as the existing 10 Xgenomics to sequence the human-mouse cell line mixed sample to obtain the detected number of the mouse single cell genes; C. distribution of sequencing read lengths (Reads) of the mouse single cell genes in different regions of a reference genome in sequencing results; D. and (3) a statistical graph of the distribution uniformity of the sequencing read length of the mouse single cell gene on the 5'-3' of the reference gene.

FIG. 7 shows the results of sequencing a pooled sample of human and murine cell lines using the method of the present invention. Wherein A is cell morphology under a microscope, B is library size distribution range, C is sequencing cell read and UMI quantity distribution, and D is human mouse cell UMI pollution distribution statistics in sequencing results.

FIG. 8 shows the results of sequencing a paraffin-embedded tissue (FFPE) sample of mouse liver tissue using the method of the present invention; wherein, A, mouse liver tissue cell nucleus after dissociation under microscope; B. performing reverse transcription reaction on the cell nucleus of the mouse liver tissue under a microscope; amplification curve of qpcr experiment; D. a nucleic acid gel electrophoresis image; E. number of gene detected in mouse liver tissue cell nucleus.

FIG. 9 is the result of the preparation of tobacco cell nuclei for sequencing using the method of the present invention. A. Plant cell nuclei dissociated under a microscope; B. plant cell nucleuses after the reverse transcription reaction under a microscope; amplification curve of qpcr experiment; D. nucleic acid gel electrophoresis image.

FIG. 10 shows the results of sequencing preparations of Chlamydomonas and blue algae samples using the method of the present invention. A. Degrading the chlamydomonas and the blue algae after the cell wall under a microscope; B. performing reverse transcription reaction on the chlamydomonas and the blue algae under a microscope; qPCR experimental amplification curve; D. nucleic acid gel electrophoresis image.

FIG. 11 shows the results of sequencing preparations for different types of fixative-immobilized cell samples using the method of the invention; A. carrying out reverse transcription on the reacted 3T3 cells under a microscope; qPCR experimental amplification curve; C. nucleic acid gel electrophoresis image.

FIG. 12 shows the results of sequencing preparations of cell samples with different capture adaptor fragments added to the cDNA ends using the method of the present invention; wherein CT values for a. qpcr experiments; B. nucleic acid gel electrophoresis image.

FIG. 13 shows the results of sequencing a mouse brain tissue sample using the method of the present invention. Where A is the gene count, read number and mitochondrial gene distribution obtained by sequencing the samples using protocol one and protocol two. B is a grouping of brain tissue and a high expression gene heatmap for both approaches. C is a correlation analysis of brain tissue clustering for both methods.

Detailed Description

The embodiment of the invention provides a single-cell transcriptome whole RNA sequencing method (as shown in figure 2), which comprises the following specific steps: pretreating a cell sample; performing in-situ reverse transcription reaction on the fixed single cells by combining target RNA by using a reverse transcription primer, wherein the reverse transcription primer comprises a tag sequence; then adding a capture linker at the tail end of the first cDNA strand synthesized by reverse transcription; subsequently creating a single chamber containing a single encoded microsphere, single cells and reactive reagents; combining the coded microspheres with the first cDNA through the complementary strand of the capture joint in a single chamber, and performing extension reaction to synthesize a second cDNA strand with a bar code label; and finally, carrying out PCR amplification and high-throughput sequencing on the obtained double-stranded cDNA. The sequencing methods of the present application allow for the formation of a single chamber containing two or more single cells in a cell isolation step, thereby creating a high throughput, high sensitivity single cell transcriptome sequencing platform.

Definition of

Unicellular cells herein include, but are not limited to, unicellular/mononuclear cells of eukaryotic cells, prokaryotes (bacteria, actinomycetes, rickettsia, chlamydia, mycoplasma, cyanobacteria, archaea, and the like), unicellular algae, viruses, and the like.

The cell sample herein includes, but is not limited to, a fixed sample of cells, a cryopreserved sample, a paraffin embedded sample (FFPE), and the like.

RNA herein includes coding RNA and various forms of non-coding RNA that can be detected, such as miRNA, lncRNA, siRNA, circRNA, and the like.

The different types of cells herein may be cells of different species, or may be cells of the same species (e.g., cells of different culture batches, cells of different origin).

The reverse transcription primer of the present invention comprises at least a tag sequence and an RNA-binding sequence that binds to a target RNA sequence, and the RNA-binding sequence may be a random RNA-binding sequence, or an RNA-binding sequence designed for a specific gene, or a combination thereof, and in practice, a Poly (dT) primer may be added to the reverse transcription primer as appropriate. The tag sequence may distinguish between different cells when reading the sequencing data, it may be a barcode fragment or other base sequence that can distinguish between different cells. In addition, the reverse transcription primer may also contain a downstream amplification primer complement fragment to allow complementary binding to the downstream amplification primer in a subsequent PCR amplification reaction, if desired. In the embodiment of the present invention, the reverse transcription primer comprises, in order from 5 'to 3', a downstream amplification primer complementary fragment, a tag sequence and 6 random base sequences (i.e. 5 '-downstream amplification primer complementary fragment amplification-tag sequence-NNNNNN N-3', N ═ dG, dA, dT or dC), wherein the 6 random base sequences can bind to a target RNA sequence, and the downstream amplification primer complementary fragment is used for binding to a downstream amplification primer in a PCR amplification reaction. For the sake of distinction, the "reverse transcription random primer" in the present application is also a primer that can be used in the reverse transcription step, which is distinguished from the reverse transcription primer by not containing a tag sequence, and in the present embodiment the reverse transcription random primer is composed of, in order from 5 'to 3', a downstream amplification primer complementary fragment and 6 random base sequences (i.e., 5 '-downstream amplification primer complementary fragment-NNNNNN-3', N ═ dG, dA, dT or dC), wherein the 6 random base sequences can bind to a target RNA sequence, and the downstream amplification primer complementary fragment is used to bind to a downstream amplification primer in a PCR amplification reaction.

The Barcode (Barcode) fragment herein refers to a string of base sequences for differentiating different cells, and is required to be stable, synthesizable, and highly variable as a cell tag. The appropriate barcode fragment can generally be designed by itself or can be selected in a barcode library. Two types of single cell sequencing technologies, 10 Xgenomics and new cell barcode libraries, currently on the market can be found in the open-source quantitative software Cellanger and CeleSCope, respectively.

Hereinafter, the main steps in the sequencing method of the present invention will be described in general.

Preparation of encoded microspheres

The single-stranded DNA on the encoded microspheres of the present invention consists of the upstream amplification primer complementary fragment, the barcode, the UMI (unique multiplex index), and the capture adaptor complementary strand (see FIG. 3). Combining the complementary segment of the upstream amplification primer with the upstream amplification primer in a PCR amplification reaction; the barcode is used to label the cDNA in the same cell partition; UMI is a random sequence used to label each original cDNA; the complementary strand of the capture linker is bound to the capture linker attached to the first cDNA strand in a second strand cDNA synthesis reaction. The encoded microspheres in embodiments of the invention can be used as microspheres in a single cell sequencing kit from 10 XGenomics, 1CellBio, New Gegen, BD Rhapbody, and the like.

Sample pretreatment

Preparing single cell suspension: corresponding digestive enzymes can be selected corresponding to different types of cell samples so as to prepare the cell samples into single cell suspensions. For example, the cultured cells may be digested into single cells with trypsin/EDTA; fresh tissue can be digested by corresponding digestive enzymes (such as collagenase I and dispase selected from muscle tissue and collagenase IV selected from liver tissue), and then filtered and washed to prepare single cells; for freezing and storing a sample, quickly melting the sample in a water bath kettle at 25-60 ℃; for a paraffin embedded sample (FFPE), xylene or other environment-friendly dewaxing agents are used for dewaxing, and then decrosslinking is carried out; for sequencing of single-cell nuclear transcriptome, a single-cell sample is treated with a strong nonionic surfactant (NP-40, etc.) to lyse the cell membrane.

Fixing single cells: when the sample to be detected is of a single cell type, fixing the single cell suspension of the cell sample to be detected by using the fixing liquid. When the sample to be detected contains different types of cells, the single cell suspensions of the cell samples to be detected of all types can be respectively fixed by using a fixing solution and then mixed, and then the subsequent reverse transcription is carried out; or mixing cell samples to be detected to prepare single cell suspension, fixing the single cell suspension by using a fixing solution, and then carrying out subsequent reverse transcription; or the single cell suspension of each type of cell sample to be detected can be respectively fixed by using a fixing solution, and the samples of different types of cells are mixed after the subsequent reverse transcription and before the cells are separated. Generally, a fixing solution is used to process a sample, and the structure of macromolecules (RNA, protein, etc.) inside the cell/nucleus is fixed, so that the macromolecules maintain the intact morphology, structure and composition of the single cell/nucleus in the subsequent experiment process, and the RNA can be stably fixed in the cell/nucleus. During operation, proper fixative may be selected based on the characteristics of different sample types, including but not limited to pure fixative, such as paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, mercury bichromate, etc., and mixed fixative, such as acetic acid-alcohol mixed solution, formalin-acetic acid-alcohol solution, Dungen's fixative, etc. And different fixed times, for example 15min to 30min or overnight.

Reverse transcription

In situ reverse transcription reaction takes place on the RNA of the fixed cells by multi-site binding of primers for the reverse transcription, which can be selected according to different practical requirements. When the primers added are reverse transcription random primers that do not include a tag sequence, to avoid contamination, only one single cell is contained in a single chamber after cell separation. When the primers added are reverse transcription primers comprising tag sequences, the separation of the cells allows at least part of the individual chambers to contain two or more cells, for example reverse transcription in a 96-well plate, where reverse transcription is performed in different wells, where a reverse transcription primer carrying a specific tag sequence is added to each well, and subsequently cells carrying different tag sequences in the cell separation step may be separated into the same cell separation.

10 percent TritonX-10 can be added into the reverse transcription system to play a role in permeating the cell membrane of bacteria, so that a reaction reagent can more easily enter the bacteria.

Add and catch joint

Adding a reverse transcription primer to the 3' tail end of the first cDNA strandCapture adapterSo that it can be combined with the single-stranded cDNA of the encoded microsphereCapture adaptor complementary strandBinding to further synthesize the second strand of cDNA. In embodiments of the invention, the first cDNA strand may be terminated by end transferPoly (dA), Poly (dT), Poly (dG) or Poly (dC)Capture Joint(ii) a Or adding a specific capture joint at the tail end of the first cDNA chain by a DNA connection method; alternatively, a template displacement method may be used in which a reverse transcriptase is used to add three dC to the tail of the first cDNA strand after reverse transcription in the reverse transcription stepFishing device Connector. In contrast, on the encoded microsphere single stranded cDNACapture adaptor complementary strandCan be adjusted accordingly.

Cell separation

Cell separation refers to the formation of a single chamber comprising a single encoded microsphere, one or more single cells, and reagents. To complete the extension reaction for the subsequent synthesis of the second strand of the cDNA, the extension reaction reagents in the single chamber typically include a DNA polymerase, dNTPs and a reaction buffer. Different microfluidic chips can be designed to generate micro-droplets to separate cells or a microplate (microwell) technology is used to separate cells according to the sizes and types of different cells. Typically, a corresponding number of single droplets are collected or a corresponding number of microwells are prepared, depending on the number of cells to be detected for sequencing. Finally, the number of the single cells in the single cavity is related to the concentration of the single cells in the system in the cell separation process, when the cell concentration is gradually increased, the proportion of the empty liquid drops after the cell separation is gradually reduced, and when the cell concentration reaches a certain value, two or even more than two single cells are contained in the single cavity after the cell separation. In particular, compared with the cell separation system in which a single chamber contains only one single cell after cell separation, the cell concentration in the cell separation system in the method of the present invention can be increased by 10-20 times, and at this time, the proportion of empty droplets after cell separation will be greatly reduced, and part of the single chamber will contain two or even more single cells.

Synthesis of the second strand of cDNA

The complementary strand of the capture linker on the single stranded cDNA encoding the microsphere linkage is bound to the capture linker attached to the first strand of the cDNA, which is subsequently extended by DNA polymerase to synthesize the second strand of the cDNA. The second strand of the resulting cDNA contains the upstream amplification primer complement, barcode, UMI, capture adapter complement, and cDNA sequence.

Construction of libraries and high throughput sequencing

Firstly, original double-stranded cDNA after the extension reaction in the last step is purified by a magnetic bead method, and an upstream primer, a downstream primer and a reagent for PCR amplification reaction are added to carry out PCR amplification on the original double-stranded cDNA. The PCR amplification product is purified by the magnetic bead method, and sequencing adapters (adapters) are attached to both ends of the PCR amplification product, for example, a TA cloning adapter library method or a PCR method can be used. The constructed library can be subjected to high-throughput sequencing by using an Illumina sequencing platform or a Huada Ching sequencing platform. The upstream primer and the downstream primer of the PCR amplification reaction are designed and synthesized according to the complementary segment of the upstream amplification primer on the coding microsphere single-stranded cDNA and the complementary segment of the downstream amplification primer in the reverse transcription random primer respectively.

Detailed Description

In order to make the objects, 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 with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention.

All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.

Example 1: sequencing of E.coli samples (Single cell contained in Single Chamber after cell isolation)

Sample pretreatment

Respectively taking about 100 ten thousand different strains (1, 2, 3 and 4) of escherichia coli to verify that the escherichia coli strain can be used for sequencing a single-cell transcriptome of a bacterial sample, adding PBS (PBST) containing 0.05% Tween-20 (purchased from Shanghai Biotechnology Co., Ltd.) to wash for three times, removing agglomerated bacteria through shaking and filtering to enable the bacteria in the sample to form a single-bacteria suspension, adding 1% paraformaldehyde (purchased from Beijing Solebao science and technology Co., Ltd.), and standing at 4 ℃ for overnight fixation. The fixed bacterial specimens were washed three times with PBST buffer, added with lysozyme (available from Thermo Fisher Scientific, USA) to lyse the cell walls, treated at 37 ℃ for 15 minutes, and washed three times with PBST buffer.

Reverse transcription

The pretreated specimen was subjected to reverse transcription reaction using a reverse transcription-permeation reagent containing reverse transcriptase (available from Thermo Fisher Scientific, usa), reverse transcription buffer, dNTPs (available from beijing solibao science and technology ltd), reverse transcription random primer (synthesized by shanghai bio-bio ltd), and 10% triton x-10 (available from shanghai bio-bio ltd), and every 50ul of the reverse transcription reaction system was used for every 50 million bacteria. After the reaction is finished, PBST buffer solution is added for washing three times.

Add and catch joint

After reverse transcription, terminal transferase (available from Thermo Fisher Scientific, USA), dCTP (available from Shanghai Biotech, Ltd.) and reaction buffer were added to the sample, and incubated at 37 ℃ for 30 minutes to add Poly (dC) capture linker fragment to the 3' end of the first cDNA strand. After the reaction is finished, PBST buffer solution is added for washing three times. Microscopic examination showed that the E.coli sample remained in a dispersed, intact single bacterial form after cell wall lysis and reverse transcription (see FIGS. 4A-B).

Cell separation

Counting each sample, and adjusting the bacterial concentration to 200-400/ul. The bacterial sample, the extension reaction reagent (including DNA polymerase (available from Thermo Fisher Scientific, USA), dNTPs and reaction buffer), the encoding microsphere (available from CellBio, USA), and the oil phase (containing 0.2% of surfactant, electron fluoridizing solution 7500, available from 3M, USA) are added into the syringe, and connected to the corresponding liquid inlet of the microfluidic chip through flexible tubes, respectively, and set the appropriate flow rate, to form a single water-in-oil droplet containing a single bacterium, a single encoding microsphere, and the extension reaction reagent, and to collect about 200ul of single droplet, to form a single chamber containing a single cell separation.

Second strand of synthetic cDNA

The single collected droplets were dispensed into different tubes and then subjected to an extension reaction to synthesize a barcode-labeled second strand of cDNA in the single droplet. After the extension reaction was completed, 20% PFO (20% of 1H,1H,2H,2H-Perfluorooctanol in E-fluroin 7500, Sigma-Aldrich, USA) was added to each tube to break up single droplets, and cDNA in the extraction tubes was purified by the magnetic bead method (magnetic beads, Beckman Coulter, USA).

Construction of libraries and high throughput sequencing

A qPCR experiment is carried out by taking partial double-stranded cDNA (200-400 cells) as a template to detect the content of the captured total cDNA, and the result shows that the obtained CT value is about 17 (see figure 4C). The appropriate number of amplification cycles is calculated from the CT values obtained (CT values plus 3 cycles are typically taken) and the cDNA samples in the remaining tubes are further subjected to PCR amplification reactions to amplify the encoded cDNA to the appropriate total content (about 100 ng). 3ul of amplification products are taken for nucleic acid electrophoresis, and the result shows that dispersed strips are obtained, the size distribution of the strips is mainly 200-500bp (see figure 4D), which indicates that the cDNA captured from the Escherichia coli sample by the method has high capture rate and wide coverage range, and can be used for subsequent high-throughput sequencing.

100ng of the amplified cDNA was subjected to end repair and A-tailing by TA cloning ligation linker library construction, and a linker was ligated (library construction kit available from Illumina, USA). The constructed library was subjected to high throughput sequencing using the Illumina sequencing platform. Sequencing results show that the number of genes distributed in 1000-3000 (see figure 4E) can be detected in a single escherichia coli, and all the genes expressed in the escherichia coli are basically contained, so that the method can be applied to sequencing of the single-cell transcriptome of the bacteria and is high in sensitivity.

Example 2: coli and B.subtilis mixed sample sequencing (only contained in a single chamber after cell isolation) One cell)

About 100 ten thousand mixed samples of escherichia coli and bacillus subtilis are taken to verify the accuracy of single cell transcriptome sequencing by applying the method. Sample pretreatment, reverse transcription, addition of capture linker, cell separation, second strand cDNA synthesis, library construction and high throughput sequencing were performed as described in example 1 above. The sequencing results showed that after genome alignment, a total of 251 bacteria were detected in the mixed samples of E.coli and Bacillus subtilis, of which 4 bacteria contained both E.coli cDNA and Bacillus subtilis cDNA, which was usually caused by the two bacteria entering the same droplet, and the remaining bacteria contained almost only E.coli cDNA or Bacillus subtilis cDNA, with an overall contamination rate of approximately less than 2% (see FIG. 5). The application of the invention to single cell transcriptome sequencing has high accuracy, can well separate escherichia coli and bacillus subtilis mixed in a sample, and has less cross contamination among species.

Example 3: sequencing of mixed samples of human and mouse cell lines

Sequencing of mouse 3T3 cells alone(Single cell contained in Single Chamber after cell isolation): about 100 million human-mouse cell line mixed samples (50% human HEK293 cells and 50% mouse 3T3 cells) frozen at-80 ℃ were taken, frozen cell samples were taken out and rapidly thawed in a 37 ℃ water bath, washed three times with PBS buffer, added with 1% paraformaldehyde, and left to stand at 4 ℃ for overnight fixation. The fixed cell cryopreserved sample is added with PBST buffer solution for three times of washing. Reverse transcription, addition of capture linker, cell separation, second strand cDNA synthesis, library construction and high throughput sequencing were then performed as described in example 1 above. The data of the mouse 3T3 cells are separated after genome comparison, and the result shows that the method can detect the number distribution of mouse single cell genes in 4000-10000 (see figure 6A) in a frozen cell sample; the literature Systematic diagnosis of single-cell and single-nuclear RNA-sequencing methods (Systematic comparison of Single-cell and Single-Nuclear RNA sequencing methods, Jianrui Ding et al, Nat Biotechnol.2020 June; 38(6): 737-In the section), seven prior art single cell transcriptome sequencing methods (seven sequencing methods including 2 microplate-based low-pass methods (Smart-Seq2 and CEL-Seq2) and 5 high-pass methods (10X Chromium, Drop-Seq, Seq-Well, inDrops and sci-RNA-Seq)) were reported to sequence human and mouse cell line mixed samples (50% human HEK293 cells and 50% mouse 3T3 cells), and compared with the sequencing data of mouse single cells detected by the seven methods, the sensitivity of the sequencing method of the invention reaches the low-pass single cell sequencing method such as Smart-Seq2 and the like under the same sequencing depth, and the high-throughput sequencing method far higher than 10X Genomics (10X Chromium) (see fig. 6B). Sequencing comparison results show that sequencing read lengths of the mouse single cells are distributed in different regions of a reference genome, including coding RNA, regions between fund, introns and untranslated regions (see figure 6C), and the sequencing method can be used for sequencing a whole transcriptome; sequencing alignment showed that sequencing reads of mouse single cells were evenly distributed on 5'-3' of the reference gene (see figure 6D).

Simultaneous sequencing of human HEK293 cells and mouse 3T3 cells(in at least part of a single chamber after cell division Comprising two or more single cells): about 500 million human-mouse cell line mixed samples (50% human HEK293 cells and 50% mouse 3T3 cells) frozen at-80 ℃ were taken, the frozen cell samples were taken out and rapidly thawed in a 37 ℃ water bath, washed three times with PBS buffer, added with 1% paraformaldehyde, and left to stand at 4 ℃ for overnight fixation. The fixed cell cryopreserved samples were washed three times with PBST buffer (see fig. 7A). Reverse transcription, addition of capture linker, cell separation, second strand cDNA synthesis, library construction and high throughput sequencing were then performed as described in example 1 above. The reverse transcription process is carried out in a 96-well plate, the reaction system of each well is reduced to 10ul, and the primers used in each well are reverse transcription primers carrying different tag sequences, so that the cDNA of cells in the same well carries the same tag sequence, and the cDNA of cells in different wells carries different tag sequences. When the cells are separated, 10 times of cell samples (2000-4000 per ul) are used, the concentration of other reagents and microspheres is unchanged, and a single cavity is formedThe chamber contains a plurality of single-cell partitions. The library size was controlled at 200-500bp (see FIG. 7B). The data of mouse 3T3 and human 293 cells are separated after genome comparison, and the result shows that the method can detect more cells in a human-mouse mixed experiment by a pre-labeling scheme, the pollution rate is controlled at a lower level (1.2%), and the number of detected transcripts can reach about 6000 (see figures 7C-D).

Example 4: mouse liver tissue paraffin-embedded tissue (FFPE) sample sequencing preparation (Single Chamber after cell isolation) In which only one single cell is included)

About 20mg of a paraffin-embedded sample of mouse liver tissue is taken, firstly, the paraffin-embedded sample is dewaxed and rehydrated, cell lysate is added to crack the tissue into single cell nuclei, and PBST is added to wash the single cell nuclei for three times. Reverse transcription, addition of capture linker, cell separation, synthesis of second strand cDNA, library construction and high throughput sequencing were then performed as described in example 1 above. The results show that single dispersed nuclei successfully isolated from FFPE samples can retain the dispersed, intact single nucleus morphology after reverse transcription reaction (see fig. 8A, B). After obtaining the purified double-stranded cDNA product, taking partial cDNA (about 100 cell nuclei) as a template to carry out qPCR experiment to detect the content of the captured total cDNA, and obtaining a CT value of about 10 (figure 8C), which shows that the method has higher cDNA capturing rate in the single cell nucleus of the FFPE sample. PCR amplification (15 cycles) is carried out on the cDNA product according to the CT value, 3ul of amplification product is taken for nucleic acid electrophoresis, and a dispersed strip is obtained, wherein the size distribution of the strip is mainly 200-500bp (figure 8D), which shows that the method has high capture rate and wide coverage range of the cDNA captured in the single cell nucleus of the FFPE sample, and can be used for subsequent sequencing. The sequencing result shows that 2000-3000 genes can be detected in a single cell nucleus of the FFPE sample by the method (see figure 8E), which indicates that the method can be applied to the sequencing of the single cell nucleus transcriptome of the FFPE sample.

Example 5: tobacco nuclear sample sequencing preparation (Single cell contained in single chamber after cell separation)

Fresh tobacco samples were dissociated into single cell nuclei using a single cell preparation instrument (see FIG. 9A), and about 100 million samples of tobacco cell nuclei were taken, washed three times with PBS buffer, added with 1% paraformaldehyde, and fixed overnight at 4 ℃. After fixation, tobacco cell nuclear samples were washed three times with PBST buffer. The library was then constructed by reverse transcription, addition of capture linker, cell separation, second strand cDNA synthesis, and reference to the methods described in example 1 above. The results show that the tobacco nuclear samples can still maintain the dispersed and intact mononuclear morphology after the reverse transcription reaction (see fig. 9B). The CT value of the tobacco cell nucleus obtained from the qPCR experiment result is about 16 (see figure 9C), which shows that the method of the invention can capture more cDNA in the tobacco cell nucleus sample. The nucleic acid gel electrophoresis result obtains a dispersive strip, the size distribution of the strip is mainly 150-300 bp (figure 9D), and the result shows that the cDNA captured from the single cells of the tobacco cell nucleus sample by the method has high capture rate and wide coverage range, and can be used for subsequent sequencing.

Example 6: sequencing preparation of Chlamydomonas and blue algae mixed sample (after cell separation, only one single cell is contained in a single chamber Cell)

Respectively take about 100 ten thousandChlamydomonas and cyanobacteria samplesVerification that the invention patent can be used forChlamydomonas and blue algaeSingle cell transcriptome sequencing of the samples. Adding PBS buffer solution into the chlamydomonas and blue algae samples for washing three times, adding 1% paraformaldehyde, and standing at 4 ℃ for fixing overnight. And after the fixation is finished, washing the chlamydomonas and blue algae samples for three times by using PBST buffer solution. For Chlamydomonas samples, cell wall enzymatic hydrolysate containing 4% cellulase, 0.5% pectinase, 0.3mol/L mannitol and pH 6.5 was added, treated at 37 ℃ for 15 minutes, and washed three times with PBST buffer. For blue algae samples, lysozyme was added to lyse cell walls, treated at 37 ℃ for 15 minutes, and washed three times with PBST buffer. The cell wall lysed Chlamydomonas and cyanobacteria samples were then reverse transcribed, linker captured, cell separated, cDNA second strand synthesized, and library constructed as described in example 1 above. The results showed that the Chlamydomonas and blue algae samples maintained a dispersed, intact single cell morphology after cell wall lysis and reverse transcription (see FIG. 10A, B). qPCR test resultsThe CT value of the chlamydomonas sample is about 17, and the CT value of the blue algae sample is about 17 (figure 10C), which shows that the method has higher cDNA capturing rate on the chlamydomonas sample and the blue algae sample. The nucleic acid gel electrophoresis result obtains a dispersive strip, the size distribution of the strip is mainly 200-500bp (figure 10D), and the result shows that the cDNA captured from the chlamydomonas and blue algae sample single cells by the method has high capture rate and wide coverage range, and can be used for subsequent sequencing.

Example 7: samples of the mouse 3T3 cell line were prepared for sequencing (using different fixative solutions and different capture linkers, the single chamber only contains one single cell after the cell separation

The effect of applying different fixative solutions to the samples was verified by taking 4 groups of cultured mouse 3T3 cell lines. Pancreatin is added into a cell culture dish and digested for 3 minutes at 37 ℃, the cell is digested into single cells, PBS buffer solution is added for washing three times, 1% paraformaldehyde, 70% ethanol, acetone and 1% acetic acid (sample numbers are 1, 2, 3 and 4) are respectively added, the mixture is placed at 4 ℃ for fixation overnight, and PBST is added for washing three times after fixation is finished. The library was then constructed by reverse transcription, addition of capture linker, cell separation, second strand cDNA synthesis, and reference to the methods described in example 1 above. The results show that the cell samples fixed by different types of fixative can maintain the complete single cell morphology after reverse transcription reaction (see FIG. 11A). The method has high cDNA capture rate on cell samples fixed by different types of stationary liquids (see figure 11B), obtains dispersed strips, and has the size distribution of mainly 200-500bp (see figure 11C), which shows that the method has high cDNA capture rate and wide coverage range on the cell samples fixed by different types of stationary liquids, and can be used for subsequent high-throughput sequencing.

In addition, 7 groups of cultured mouse 3T3 cell lines were separately used to verify the effect of adding different capture linkers at the tail end of the first strand of cDNA. Pancreatin was added to the cell culture dish and digested at 37 ℃ for 3 minutes into single cells, washed three times with PBS buffer, added with 1% paraformaldehyde, fixed overnight at 4 ℃ and washed three times with PBST after fixation. Samples of 6 groups of fixed cells were subjected to reverse transcription reaction according to the method described in example 2 above. After 4 groups of cells were reverse transcribed, dATPs, dTTPs, dGTPs and dCTPs, as well as terminal transferase and reaction buffer were added to each of the samples, and the samples were incubated at 37 ℃ for 30 minutes, and Poly (dA), Poly (dT), Poly (dG) and Poly (dC) were added to the cDNA ends as capture linker fragments. And adding DNA ligase, a specific capture adaptor fragment and a reaction buffer solution into another 2 groups of cell samples after the reverse transcription is finished, incubating for 30 minutes at 37 ℃, and adding the specific capture adaptor fragment to the tail end of the cDNA. And adding reverse transcriptase (Moloney Murine Leukamia Virus) into 1 group of cell samples after fixation, reverse transcription random primers, dNTPs, reverse transcription reaction buffer solution and a reverse transcription-permeation reaction reagent containing 10 percent TritonX-10 to perform reverse transcription reaction, and adding three dC (dC) as capture joint fragments at the tail end of the cDNA after the reverse transcription is finished. The cell sample with the capture linker was subjected to cell separation, second strand cDNA synthesis, and library construction according to the methods described in examples 4, 5, and 6 above. The results show that the range of the CT value of the cell samples with different capture linker fragments is 12-13, which indicates that more cDNAs can be captured in the cells by using different capture linker fragments in the method of the present invention (see FIG. 12A), and the gel electrophoresis results show that dispersed bands are obtained, the size distribution of the bands is mainly 200-500bp (see FIG. 12B), which indicates that the cDNAs captured in the cells by using different capture linker fragments in the method of the present invention have high capture rate and wide coverage range, and can be used for subsequent high-throughput sequencing.

Example 8: mouse brain samples were prepared for sequencing (comparison of different reverse transcriptions and cell separation systems)

The first scheme is as follows: separating brain tissue after neck breaking and killing of mouse, crushing brain tissue with liquid nitrogen, and cracking tissue with cracking liquid Powder ofDissociated into single nuclei. About 600 ten thousand mouse brain cell nucleus samples were taken, washed three times with PBS buffer, added with 1% paraformaldehyde, and fixed overnight at 4 ℃. After fixation, the nuclear samples were washed three times with PBST buffer. 100 ten thousand mouse nuclei were subjected to reverse transcription, addition of a capture linker, cell separation, second strand cDNA synthesis, and library construction according to the method described in example 1 above, whereinThe transcription step uses reverse transcription random primer without label sequence, and the cell separation forms single chamber containing single cell.

Scheme II: taking 500 ten thousand mouse cell nuclei, carrying out reverse transcription, adding a capture linker, separating cells, synthesizing a cDNA second chain and constructing a library by referring to the method described in the above embodiment 1, wherein the reverse transcription process is carried out in a 96-well plate, compared with the scheme I, the reaction system of each well is reduced to 10ul, a primer used in each well is a reverse transcription primer carrying a label sequence, a cell sample (2000-4000 per ul) with 10 times concentration is used for separating cells, the concentration of other reagents and microspheres is unchanged, at least part of single chambers after the cells are separated contain a plurality of single cells, and the number of empty liquid drops is reduced.

As can be seen from the figure, under the condition of collecting the same number of droplets, the second protocol obtained more cell numbers, and the number of genes and the grouping condition detected at the same time were highly consistent with the first protocol (FIG. 13). This shows that the method of the present invention can not only increase the cell flux in a single measurement, but also increase the efficiency of cell capture and reduce the cost by adding the tag sequence in the reverse transcription process.

The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. A sequencing method of a single cell transcriptome is characterized in that a cell sample to be detected is prepared into a single cell suspension and then is fixed by a fixing solution; and using reverse transcription primer to make in-situ reverse transcription reaction on the fixed single-cell RNA to synthesize cDNA first chain;

wherein the reverse transcription primer comprises a tag sequence.

2. The method of claim 1, wherein the reverse transcription primer comprises a tag sequence and an RNA binding sequence that is a random RNA binding sequence, an RNA binding sequence designed for a target RNA sequence, or a combination thereof.

3. The method of claim 1 or 2, further comprising adding a capture linker to the end of the first strand of the cDNA from the reverse transcribed single cell, wherein the capture linker is complementary to the sequence encoding the complementary strand of the capture linker on the microsphere; the capture adaptor is any fragment of known sequence, preferably the capture adaptor is a Poly (dA) fragment, a Poly (dT) fragment, a Poly (dG) fragment or a Poly (dC) fragment.

4. The method of any one of claims 1-3, further comprising containing a single cell and a single said encoded microsphere in a single chamber to form a cell partition; and synthesizing a second strand of cDNA in the single chamber after forming the cell partition;

wherein two or more single cells are included in at least a portion of the single chamber after forming the cell partition.

5. The method of claim 4, wherein the two or more single cells are cells of the same type or different types.

6. The method of claim 4 or 5, further comprising PCR amplification and pooling and sequencing of the double stranded cDNA after synthesis of the second strand of the cDNA.

7. The method of any one of claims 3-6, wherein the method uses a single-stranded DNA on the encoded microspheres comprising the upstream amplification primer complementary fragment, the barcode, the UMI, and the capture adaptor complementary strand; preferably the barcode is one or more barcodes; more preferably three barcodes.

8. The method of any one of claims 1 to 7, wherein the cell separation is performed using a microfluidic chip or a microplate.

9. The method of any one of claims 2-8, wherein the fixative solution is a simple fixative solution or a mixed fixative solution; preferably the simple fixative solutions include, but are not limited to, paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, and mercuric chloride; preferably, the mixed fixative includes, but is not limited to, acetic acid-alcohol mixed solution, formalin-acetic acid-alcohol solution, and Bovins fixative.

10. Use of the method of any one of claims 1-9 for whole transcriptome sequencing of a single cell, a single cell nucleus, a single microorganism; preferably the use is in the fields of microbiology, basic medicine, clinical medicine, agriculture, cell biology, immunology, developmental biology, pathology, neurobiology and development, genetics, stem cells, tumors, reproductive health, metagenomics and micro-ecology, new drug development.

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