CN116287115A

CN116287115A - Method for obtaining single cell or single cell nucleus sample, sample obtained by using same and application

Info

Publication number: CN116287115A
Application number: CN202310156589.3A
Authority: CN
Inventors: 王永成; 徐子叶
Original assignee: Hangzhou Yuezhen Biotechnology Co ltd
Current assignee: Hangzhou Yuezhen Biotechnology Co ltd
Priority date: 2022-12-14
Filing date: 2023-02-23
Publication date: 2023-06-23

Abstract

The invention provides a method for obtaining a single cell or single cell nucleus sample, a cell or cell nucleus obtained by using the method and application of the cell or cell nucleus in transcriptome sequencing.

Description

Method for obtaining single cell or single cell nucleus sample, sample obtained by using same and application

Technical Field

The invention belongs to the field of biotechnology, relates to the field of single-cell sequencing, and in particular relates to a method for obtaining a single-cell or single-cell nuclear sample, a sample obtained by using the method and application of the sample.

Background

The most widely used single-cell transcriptome sequencing technology at present is a single-cell sequencing technology based on a droplet microfluidic platform developed by 10X Genomics company, the technology can realize marking, sequencing and analysis of thousands of cells, obtain a single-cell-level gene expression profile, realize division of cell subsets and detection of differential expression genes among the cell subsets, and the similar technologies are an inDrop technology and a Drop-seq technology. The single-cell transcriptome sequencing technology of the 10X Genomics platform adopts a polyT primer to capture RNA containing polyA tail in a sample, and has low detection sensitivity. And DNA double-stranded structures in samples preserved by means of freezing, refrigerating, fixing liquid, sample preservation liquid or FFPE and the like are easily damaged, and single-stranded DNA is exposed. If existing single cell transcriptome sequencing techniques are applied directly to these samples, the reverse transcription primer may simultaneously capture cDNA information from the DNA, thereby allowing a large number of intergenic regions to be detected in the sequencing results, wasting sequencing bases, and contaminating the gene expression matrix.

Disclosure of Invention

In order to solve the above-mentioned problems, in a first aspect, an object of the present invention is to provide a method for obtaining a single cell or single cell nuclear sample, the method comprising performing single-stranded DNA blocking.

In particular embodiments, the single-stranded DNA blocking comprises an in situ extension reaction on an exposed single strand of DNA in the nucleus.

In a specific embodiment, the in situ extension reaction occurs in the method of the invention by the addition of a DNA blocking reagent. In a preferred embodiment, the DNA blocking reagent comprises a DNA elongase, a blocking primer, dNTPs, and a reaction buffer. In optional embodiments, the DNA blocking reagent may further comprise Triton X-100, PEG8000, mgCl ₂ And/or MgSO ₂ 。

In a specific embodiment, the method of the invention comprises the steps of: (1) preprocessing an original sample; (2) Dissociation of the pretreated sample to obtain single cells or single nuclei; and (3) single stranded DNA blocking.

In particular embodiments, the dissociation of the pre-treated sample comprises further processing the sample to obtain single cells or single nuclei by adding proteinase K after adding a single cell dissociation solution or single nucleus dissociation solution to the pre-treated sample to dissociate the sample. In a preferred embodiment, the concentration of proteinase K is 5-15 mg/ml. In a more preferred embodiment, the treatment time is from 5 to 30 minutes.

In specific embodiments, the single cell dissociation solution is an enzyme-or chelator-containing buffer; wherein the enzyme may be selected from, but is not limited to, collagenase, neutral protease, trypsin, elastase, hyaluronidase, papain, deoxyribonuclease I; the buffer may be selected from, but is not limited to PBS, HEPES, TRIS or SSC buffers. In a preferred embodiment, the concentration of enzyme in the single cell dissociation solution is between 0.01% and 0.5% g/100ml. In a further preferred embodiment, the chelating agent is EDTA; the concentration of the chelating agent in the single-cell dissociation liquid is 0.001% -0.1% g/100ml.

In particular embodiments, the single cell nuclear dissociation solution is a buffer containing a nonionic surfactant, wherein the concentration of nonionic surfactant may be 0.05 to 0.5% (by volume), and the nonionic surfactant may be selected from, but is not limited to, NP-40, triton X-100, tween-20, tween-80, CHAPS detergent, brij-58, and Octyl Thioglucoside (OTG); the buffer may be selected from, but is not limited to PBS, HEPES, TRIS or SSC buffers.

In a specific embodiment, the single cell nuclei dissociation of the method of the invention comprises adding a nonionic surfactant to the pretreated sample followed by homogenization, shaking or ultrasonication to form a single cell nuclei suspension.

In a specific embodiment, the method of the present invention may comprise the step of fixing the single cells or nuclei by adding a fixing solution after the step (2) and before the step (3), wherein the fixing of the single cells or nuclei is performed by adding a fixing solution, and the fixing solution may be a simple fixing solution or a mixed fixing solution. In a preferred embodiment, the simple fixative may be selected from the group consisting of, but not limited to, paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, mercuric chloride, and the like. In further preferred embodiments, the mixed fixative may be selected from the group consisting of, but not limited to, acetic acid-alcohol mixtures, formalin-acetic acid-alcohol, bao Yinshi fixatives, and the like. In particular embodiments, the fixed time may be selected based on the sample being processed, and may be, for example, 15 minutes to 30 minutes or overnight.

In a specific embodiment, the method of the invention may comprise permeabilizing said single cell or single cell nucleus by adding a permeabilizing agent after the immobilization of said single cell or single cell nucleus and before step (3). In a preferred embodiment, permeabilization of the single cell nucleus can be performed by adding a buffer comprising the permeabilizing agent. In a preferred embodiment, the permeabilizing agent can be selected from the group consisting of, but not limited to, triton X-100, NP-40, saponins, tween 20, and Tween80, wherein the saponins are preferably digitalis saponins. In a more preferred embodiment, the permeabilizing agent is present in an amount of 0.1 to 0.5% (by volume); in further preferred embodiments, the buffer may be selected from the group consisting of, but not limited to, PBS, HEPES, TRIS and SSC buffers.

In particular embodiments, the original sample may be a sample including, but not limited to, a paraffin-embedded sample, a frozen sample, a chilled sample, a fresh sample, a fixative solution preservation sample, or a sample preservation solution preservation sample.

In a second aspect, the present invention aims to provide a single cell or single cell nuclear sample obtained by treatment with the method of the first aspect.

In a third aspect, the present invention aims to provide the use of the method of the first aspect or of single-cell or single-cell nuclear samples obtained by treatment according to the method of the first aspect in single-cell, single-cell nuclear, single-microorganism transcriptome sequencing; preferably the use is in the fields of microbiology, basic medicine, clinical medicine, agrology, cell biology, immunology, developmental biology, pathology, neurobiology and development, genetics, stem cells, tumour, reproductive health, metagenomics and microecology, and new drug development.

In particular embodiments, the transcriptome sequencing technique in the methods of the present invention may be single cell or single cell nuclear transcriptome sequencing techniques of a variety of different principles. Such single cell or single cell nuclear transcriptome sequencing techniques include, but are not limited to, 10X Genomics platform, BD Rhapsody platform, VASA-seq, drop-seq, smart-seq, split-pool, or single cell nuclear transcriptome sequencing techniques developed in the present laboratory (e.g., as described in chinese patent application No. 202210174619.9).

The single cell or single cell nuclear sample processed by the method is cleaner, the residual fragments on the surface of the single cell or single cell nuclear are obviously reduced, the ratio of the obtained base ratio to the intergenic region is obviously reduced when the single cell or single cell nuclear transcriptome is used for sequencing, the ratio of the obtained base ratio to the coding region, the UTR region, the intronic region and the ribosomal region is obviously increased, the utilization rate of the obtained base of the sequencing is obviously improved, and the pollution of the intergenic region to the gene expression quantity is reduced.

Drawings

FIG. 1 is a schematic diagram of a method of obtaining a single cell or single cell nuclear sample of the present invention.

FIG. 2 is a schematic diagram of a single cell or single cell nuclear transcriptome sequencing method using single cell or single cell nuclear samples obtained by the process of the present invention.

Figure 3 is a single cell nuclear micrograph of a kidney sample of FFPE isolated from a mouse at a method of the invention.

FIG. 4 is a base profile of sequencing data of single nuclei after a mouse FFPE kidney sample without single stranded DNA blocking treatment, using single stranded DNA blocking treatment and using DNase digestion treatment.

Fig. 5 is a photomicrograph of a single cell nuclear sample of FFPE kidney from mice treated with proteinase K and collagenase.

Fig. 6 is a photograph of single cell nuclear DAPI staining after FFPE tissue samples of various mice treated by the method of the invention.

FIG. 7 is a base profile of sequencing data from single nuclei following treatment of various mouse FFPE tissue samples by the method of the present invention.

FIG. 8 shows the number of single cell nuclear gene detections of various mouse FFPE tissue samples treated by the method of the present invention.

FIG. 9 is a graph showing the analysis of single cell nuclear sequencing data for a plurality of mouse FFPE tissue samples treated by the method of the present invention.

FIG. 10 shows the single cell nuclear isolation and sequencing data analysis results of FFPE samples of clinical human liver cancer treated by the method of the present invention.

FIG. 11 shows differential expression genes of cell populations of single cell nuclear sequencing data of FFPE samples of clinical human liver cancer treated by the method of the present invention.

FIG. 12 shows the single cell nuclear isolation results and sequencing data analysis results of a mouse cryopreserved heart tissue sample treated by the method of the present invention.

FIG. 13 shows single cell isolation and sequencing data analysis of fresh islet muscle tissue samples from mice treated by the method of the invention.

FIG. 14 is the results of single cell samples of fresh islet tissue from mice treated with proteinase K and the results of sequencing data analysis.

Detailed Description

Embodiments of the present invention provide a method for obtaining a single cell or single cell nuclear sample by blocking single-stranded DNA exposed in the sample by breaking double-stranded structures to avoid hybridization and extension reactions of blocking primers added during reverse transcription with these single-stranded DNA. As shown in fig. 1, the method mainly comprises pretreatment of an original sample, single-cell or single-cell nucleus acquisition of the pretreated sample and single-strand DNA blocking, wherein the single-cell or single-cell nucleus immobilization and permeabilization are optionally performed before the single-strand DNA blocking is performed, and specifically comprises the steps of: selecting a region of interest for the FFPE sample, the frozen sample, the refrigerated sample, the fixed sample or the sample stored in the sample storage liquid, taking out 5-50 mg of the sample in the region by using a puncher or cutting out a sheet larger than 50um by using a slice, and correspondingly preprocessing; adding single cell or single cell nucleus dissociation solution to dissociate to obtain single cell or single cell nucleus; optionally, continuing to add proteinase K to further treat the sample after the sample is dissociated by adding single cell or single cell nucleus dissociation solution to obtain cleaner single cells or single cell nuclei; optionally fixing and permeabilizing the single cell or single cell nucleus obtained; in-situ extension reaction is carried out on the single cell nucleus or the naked DNA single strand in the single cell nucleus, so that the naked DNA single strand is blocked.

Definition of the definition

Single cells herein include, but are not limited to, single cells of eukaryotic cells, prokaryotes (bacteria, actinomycetes, rickettsiae, chlamydia, mycoplasma, cyanobacteria, archaebacteria, etc.), single cell algae, viruses, etc., single cell nuclei herein including, but not limited to, the nuclei of the above-described cells.

The frozen sample herein refers to a human or animal or plant tissue sample or a cell sample frozen at 0 ℃ to-80 ℃ or in liquid nitrogen.

The refrigerated sample herein refers to a human or animal or plant tissue sample or a cell sample stored at 0℃to 8 ℃.

A fixed sample in this context refers to a human or animal or plant tissue sample or a cell sample stored in paraformaldehyde or formaldehyde.

The sample stored in the sample storage solution herein means a sample stored in RNAwait (non-frozen tissue RNA storage solution),

Human or animal or plant tissue samples or cell samples in various sample preservation solutions in the market such as tissue preservation solution.

FFPE samples herein refer to paraffin sections or paraffin block samples of human or animal or plant tissue samples or cell samples formed by formalin fixation, paraffin embedding.

RNA herein includes both detectable coding RNA and non-coding RNA in various forms, such as miRNA, lncRNA, siRNA, circRNA and the like.

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

The blocking primer of the present invention comprises 3 to 20Random base sequence (i.e., 5' - (NNN) _3-20 -3', n=dg, dA, dT or dC) may be randomly combined with the single stranded DNA sequence.

The detergent herein may be a buffer based on PBS, HEPES, TRIS or SSC, and 0.005 to 0.2% of a nonionic surfactant, such as Tween 20, tween80, triton X-100, etc., may be added to the detergent.

The main steps of the sequencing method employed in the examples of the present invention will be described generally hereinafter.

Sample pretreatment

The respective sample pretreatment methods may be selected for different types of samples to prepare samples of suitable size that may be dissociated into single cell suspensions without reagents affecting subsequent permeabilization, blocking, etc.

First, a sample of a suitable size may be collected, and collection methods such as scissors cutting, blade cutting, tissue microtome, punch, laser capture microdissection (LMD or LCM), micro-area sampling, etc. may be used.

The corresponding preprocessing method can then be selected for the different types of samples. For paraffin embedding

(FFPE) samples, using a dewaxing agent to remove paraffin, adding ethanol with gradient concentration for rehydration; for the frozen sample, cleaning three times by using a detergent after quick thawing; for the refrigerated samples, wash three times with detergent; removing the fixing liquid for the sample stored by the fixing liquid, and cleaning the sample three times by using a detergent; for the samples preserved by the sample preservation solution, the preservation solution is removed and washed three times with a detergent.

The paraffin removal agent for FFPE samples can be selected from xylene, dextral limonene, aqueous paraffin removal agent, environment-friendly transparent agent, hist-Clear tissue paraffin removal agent, hist-Clear II environment-friendly tissue transparent agent II, seebio nontoxic environment-friendly transparent paraffin removal agent, and the concentration gradient of ethanol for rehydration can be 100%, 98%, 95%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% and the like.

Preparation of Single cell Nuclear suspension

The corresponding single-cell nuclear dissociation method can be selected for different types of samples to prepare the samples into single-cell nuclear suspension, and the purpose is to obtain a large number of single, dispersed and nuclear-morphologically complete single-cell nuclei. And adding single-cell nucleus dissociation solution into the pretreated sample for dissociation. In order to improve the dissociation effect, the concentration of the nonionic surfactant in the dissociation liquid can be adjusted, or a plurality of different nonionic surfactants are selected, or a sample added with the dissociation liquid is homogenized by a homogenizer, or the sample added with the dissociation liquid is placed on a shaking table to vibrate, or the sample added with the dissociation liquid is subjected to ultrasonic treatment by an ultrasonic breaker. Proteinase K may then be added continuously to the sample to obtain a cleaner single cell nucleus. After the treatment, the supernatant is centrifugally removed to obtain single cell nuclei, and part of the cell nuclei are stained and observed under a microscope.

Preparation of Single cell suspension

The corresponding single cell dissociation method can be selected for different types of samples to prepare the samples into single cell suspensions, with the aim of obtaining a large number of single, dispersed, single cells with complete cell morphology. And adding single-cell dissociation solution into the pretreated sample, and incubating at 37 ℃ for digestion reaction. Proteinase K may then be added further to obtain cleaner single cells. Filtering and washing after the treatment is finished to prepare single cell suspension.

Single cell or single cell nuclear immobilization

Corresponding fixing reagents and fixing time can be selected corresponding to different types of samples, so that substances such as RNA, DNA, protein and the like in single cells or single cell nuclei can be crosslinked and fixed in the single cells or the single cell nuclei, and the single cells or the single cell nuclei can keep complete cell morphology and internal structure in the subsequent reaction steps of permeabilization, transcriptome sequencing and the like.

After the sample is prepared into single-cell nuclear suspension, adding a fixing solution for fixing, centrifuging to remove the supernatant after the fixing is finished, and washing with a detergent for three times. The operation can select proper fixing liquid according to different sample types, and the fixing liquid comprises, but is not limited to, simple fixing liquid such as paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, mercuric chloride and the like, and mixed fixing liquid such as acetic acid-alcohol mixed liquid, formalin-acetic acid-alcohol liquid, bao Yinshi fixing liquid and the like. Different fixed times may be chosen depending on the particular situation, for example 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 24 hours, etc. Different fixed temperatures may be chosen depending on the particular situation, for example 4 ℃, on ice or at room temperature, etc. For the sample of fixative solution preservation or FFPE sample, the fixation step may be omitted or the fixation time reduced.

The method of immobilization of single cells is essentially the same as that of single nuclei.

Single cell or single cell nuclear permeabilization

Corresponding permeabilization reagents and permeabilization times can be selected to break the cell membrane of a single cell or the nuclear membrane of a single cell nucleus to allow for better ingress and egress of subsequent reagents to the cell or cell nucleus. The permeabilization method of single cells is basically the same as that of single cell nuclei.

Adding a permeabilizer into the fixed sample for permeabilization, centrifuging to remove supernatant after permeabilization, and washing with a detergent for three times. During operation, a proper permeabilizing agent can be selected according to different sample types, wherein the permeabilizing agent can be selected from buffer solutions containing 0.1-0.5% (volume ratio) of Triton X-100, NP-40, saponins, tween 20, tween80, digitonin and the like, or 100 mu M Digitonin or 0.5% Saponin, and the buffer solution can be PBS, HEPES, TRIS or SSC buffer solution. According to the characteristics of different sample types, proper permeabilization time can be selected, and the permeabilization time can be 1-15 minutes.

Single stranded DNA blocking

The single-stranded DNA blocking reaction causes in-situ extension reaction on the exposed DNA single strand through the blocking primer, so that the exposed DNA single strand is blocked into double-stranded DNA, the double-stranded DNA cannot participate in subsequent reverse transcription reaction, and finally, a single-cell or single-cell nuclear sample which can be used for subsequent single-cell or single-cell nuclear transcriptome sequencing is obtained.

Adding DNA into the permeabilized single cell or single cell nucleus sampleDNA blocking reagent such as elongase, blocking primer, dNTPs, reaction buffer, etc., which can be added with TritonX-100, PEG8000, mgCl to increase blocking effect, is used for reaction at 37 deg.C for 10-90 min, and washed three times with detergent after reaction ₂ Or MgSO ₂ Etc.

Single cell or single cell nuclear transcriptome sequencing

The permeabilized sample can be sequenced using various types of single cell or single cell nuclear transcriptome sequencing techniques including, but not limited to, 10X Genomics platform, VASA-seq, drop-seq, smart-seq, split-pool, or transcriptome sequencing techniques developed by the inventors of the present application (e.g., as described in chinese patent application No. 202210174619.9) (see fig. 2).

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.

All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings.

Example 1: dissociating single cell nuclei in mouse FFPE kidney samples

The area of interest on the FFPE kidney samples of mice was selected under a microscope and about 10mg of the samples were removed from the wax block using a punch and placed in a 1.5ml centrifuge tube. 1ml of xylene was added, treated at room temperature for 5 to 10 minutes, the xylene was removed, and fresh 1ml of xylene was added again to dewax. After dewaxing, xylene is removed, and ethanol with gradient concentration of 100%, 95%, 80%, 70%, 50% and 30% is added for rehydration. After rehydration, 1ml of single cell nucleus lysis is addedThe liquid is single cell nucleus lysate containing 0.5-2% NP-20 ion surfactant and MgCl ₂ (1-5 mM) of 2 XSSC buffer solution, adding the mixture of the sample and the lysate into a Dunn homogenizer for homogenization, homogenizing for 10-30 times, standing on ice for 5-15 minutes, adding 100ul proteinase K (5-15 mg/ml) into the mixture after partial homogenization, reacting for 5-30 minutes at 25-50 ℃, centrifuging at room temperature, removing the supernatant to obtain single cell nuclei, and washing three times with a washing solution. A portion of the nuclei was stained with the nucleus specific dye DAPI and observed under a microscope. Referring to fig. 3, it can be seen that some dispersed and complete FFPE single-cell nuclei can be dissociated from the FFPE kidney samples of the mice lysed by the single-cell nuclei lysate, and the single-cell nuclei samples after DAPI staining are obviously blue under a fluorescence microscope, but still have tissue block residues (fig. 3A), and a cleaner and dispersed FFPE single-cell nuclei suspension can be obtained after filtration through a 40 μm cell sieve, and can be used for the next experiment (fig. 3B).

Example 2: single-cell nuclear permeabilization and single-strand DNA blocking

The single-cell nuclear sample of the FFPE kidney of the mice obtained in the example 1 is subjected to permeabilization, single-strand DNA blocking and single-cell nuclear whole transcriptome sequencing, and the specific method is as follows:

permeabilization

A suitable amount of the single-cell nucleus sample treated in example 1 was taken, permeabilized with a permeabilizing agent containing 0.2% Triton X-100 for 10 minutes, centrifuged to remove the supernatant after permeabilization, and washed three times with a detergent.

Single stranded DNA blocking

DNA blocking reagent containing DNA extension enzyme (1-10U/. Mu.l), blocking primer (5-50 mM), dNTPs (1-10 mM) and 1X thermo pol reaction buffer solution is added into the permeabilized sample, the reaction is carried out for 20-45 minutes at 37 ℃, and the sample is washed three times by using a detergent after the blocking is finished.

Example 3: sequencing of mouse FFPE kidney samples

Reverse transcription

Obtained in example 2Single chain DNA closureAdding reverse transcription reaction reagent containing reverse transcriptase, reverse transcription reaction buffer solution, dNTPs and reverse transcription primer into the obtained FFPE kidney single cell nuclear sample of the mice to carry out reverse transcription reaction, and adding a detergent to wash for three times after the reaction is finished.

Catch joint

The sample after reverse transcription was added with terminal transferase, dCTP and reaction buffer, and incubated at 37℃for 30 minutes. And adding a detergent to wash for three times after the reaction is finished.

Cell separation

The single cell nucleus sample, the extension reaction reagent (comprising DNA polymerase, dNTPs and reaction buffer solution), the coding microsphere and the oil phase are respectively added into an injector, are respectively connected with the corresponding liquid inlet of the microfluidic chip through hoses, and are provided with proper flow rates to form water-in-oil single liquid drops containing single cell nucleus, single coding microsphere and extension reaction reagent, the single liquid drops are collected, and the cell separation of a single chamber containing one single cell is formed.

Second Strand of cDNA Synthesis

The collected single droplets were dispensed into different tubes and then subjected to an extension reaction to synthesize a second strand of the barcode-labeled cDNA in the single droplets. After the completion of the extension reaction, the single droplet was broken, and cDNA in the extraction tube was purified by the magnetic bead method.

Construction of libraries and high throughput sequencing

And taking part of double-stranded cDNA as a template to perform qPCR experiment to detect the content of the captured total cDNA. And (3) carrying out PCR amplification on the rest cDNA, carrying out tail end repair and A tail addition on the amplified cDNA by adopting a TA cloning connection joint library construction method, and connecting an upper joint by using a library construction kit. The constructed library was subjected to high throughput sequencing using an Illumina sequencing platform.

Sequencing results show that the proportion of the intergenic regions detected by sequencing in the single-stranded DNA-blocked mouse FFPE kidney single-cell nuclear sample is obviously reduced from 61.2% to 11.3%; the corresponding coding region, UTR region, intron region, and ribosomal region ratios were all significantly increased, with the coding region ratio increasing from 1.1% to 4.0%, the UTR region ratio increasing from 1.2% to 6.9%, the intron region ratio increasing from 36.5% to 68.7%, and the ribosomal region ratio increasing from 0.0% to 9.1% (fig. 4A, B).

To eliminate the contamination of the experimental results with single-stranded DNA, we have also tried methods of digesting DNA in single cells or nuclei with dnase. The single cell nuclear sample of the FFPE kidney of the mouse obtained according to the method described in example 1 was permeabilized according to the permeabilization method described in example 2, dnase and reaction buffer were added to the single cell nuclear sample of the FFPE kidney of the mouse after permeabilization, incubation was performed for 30 to 60 minutes at 37 ℃, 1 μl of 25mm edta was added to the reaction system, incubation was performed for 10 minutes at 65 ℃ to inactivate dnase, and washing with detergent was performed three times after the reaction was completed. Reverse transcription, capture linker addition, cell separation, cDNA second strand synthesis, library construction, and high throughput sequencing were performed on mouse FFPE kidney single cell nuclear samples after DNA digestion with the methods described in example 3 above. The results show that the ratio of the intergenic region, the coding region, the UTR region, the intron region and the ribosomal region detected by sequencing in the single-cell nuclear sample of the FFPE kidney of the mice after DNA digestion is not obviously changed (figure 4C), which shows that the pollution of single-chain DNA to experimental results cannot be effectively eliminated by using a DNase digestion method.

Example 4: proteinase K treated mice FFPE kidney single cell nuclear sample

Sample pretreatment and single cell nucleus dissociation are performed on the FFPE kidney tissue samples of the mice by the method described in the example 1, 100ul proteinase K (5-15 mg/ml) is added into the mixture after partial homogenate, the reaction is carried out for 5-30 minutes at 25-50 ℃, the centrifugation is carried out at room temperature, the supernatant is removed to obtain single cell nuclei, and the single cell nuclei are washed three times by washing liquid. A portion of the nuclei was stained with the nucleus specific dye DAPI and observed under a microscope. Referring to fig. 5, it can be seen that the protease K treated mice FFPE kidney single cell nucleus samples had cleaner background, single dispersed nuclei, and maintained intact nuclear morphology (fig. 5A).

For comparison with the effect of the treatment with proteinase K above, a control experiment was also performed in which the single cell nuclei were treated with collagenase. Taking a part of the homogenized mixture, adding 100ul collagenase (5-15 mg/ml), reacting for 5-30 minutes at 25-50 ℃, centrifuging at room temperature, removing the supernatant to obtain single cell nuclei, and washing with a washing liquid three times. A portion of the nuclei was stained with the nucleus specific dye DAPI and observed under a microscope. Referring to FIG. 5B, it can be seen that the collagenase treated single cell nuclei had more cytoplasm on their surface and that some cells were still adhered together (FIG. 5B), so proteinase K had better effect on single cell nuclei dissociation than other proteases.

Example 5: sequencing of multiple FFPE tissue samples from mice

Sample pretreatment, single cell nuclear dissociation, proteinase K treatment, permeabilization, single strand DNA blocking, reverse transcription, addition of capture linkers, cell separation, cDNA second strand synthesis, library construction, and high throughput sequencing were performed on various FFPE tissue samples (including liver, kidney, heart, testis) from mice with reference to the methods described in examples 1-4 above. The results showed that intact nuclei could be isolated from various FFPE tissue samples from mice (fig. 6A-D). Sequencing results show that the base distribution ratio detected in single cell nuclei of various FFPE tissue samples of the mice is in a normal range, wherein the ratio of intergenic regions is 11.9-29.8%, the ratio of coding regions is 4.1-7.6%, the ratio of UTR regions is 5.4-6.7%, the ratio of intronic regions is 31.7-53.8%, and the ratio of ribosomal regions is 18.1-28.9% (figures 7A-D). The gene comparison result shows that the number of genes which can be detected in single nuclei of various FFPE tissue samples of the mice is 3000-4000 (figure 8). The results of the clustering analysis of the calculated single cell nuclear transcriptome expression matrices showed that cells from multiple FFPE tissue samples from mice could be clustered into multiple cell populations (fig. 9A), and cell types of these cell populations could be identified by looking at the reported marker gene expression in these cell populations (fig. 9B).

Example 6: sequencing of clinical FFPE human liver cancer tissue samples

Sample pretreatment, single-cell nuclear dissociation, proteinase K treatment, permeabilization, single-strand DNA blocking, and single-cell nuclear transcriptome sequencing were performed on clinical human liver cancer FFPE samples with reference to the methods described in examples 1-4 above. The results showed that intact nuclei could be isolated from clinical human liver cancer FFPE samples (fig. 10A). Sequencing results showed that the base distribution ratio detected in single cell nuclei of FFPE samples of clinical human liver cancer was in the normal range, in which the ratio of intergenic regions was 8.0%, the ratio of coding regions was 12.0%, the ratio of UTR regions was 19.0%, the ratio of intronic regions was 45.8%, and the ratio of ribosomal regions was 15.3% (fig. 10B). The gene comparison result shows that the number of genes which can be detected in single cell nuclei of the FFPE sample of the clinical human liver cancer is about 3000 (figure 10C), and the number of UMI is about 10000 (figure 10D). The results of the cluster analysis of the calculated single cell nuclear transcriptome expression matrix showed that cells from the clinical human liver cancer FFPE sample could be clustered into a plurality of cell populations, and cell types of these cell populations could be identified based on the reported expression of the marker genes in these cell populations, including most cell types in human liver tissue (hepatocyte 1, hepatocyte 2, hepatocyte 3, hepatocyte macrophage, hepatic satellite cell, T cell, B cell) (fig. 10E). By calculating the differentially expressed genes between different cell populations, new marker genes can be identified (FIG. 11).

Example 7: sequencing of mouse cryopreserved cardiac tissue samples

Quickly thawing a mouse frozen heart tissue sample, selecting a region of interest on the mouse frozen heart tissue sample under a microscope, taking about 10mg of the sample out by a puncher, placing the sample into a centrifuge tube, adding a precooled washing agent, and washing for three times. 1ml of single cell nucleus lysate (containing 0.5-2% NP-20 nonionic surfactant and 1-5 mM MgCl) is added ₂ Adding the mixture of the sample and the lysate into a Dunn homogenizer for homogenization for 10-30 times, standing on ice for 5-15 minutes, adding the mixture of the sample and the lysate into the Dunn homogenizer for homogenization for 10-30 times, standing on ice for 5-15 minutes, adding 100ul proteinase K (5-15 mg/ml) into the homogenized mixture, reacting for 5-30 minutes at 25-50 ℃, centrifuging at room temperature, and removingThe supernatant yielded single nuclei, which were washed three times with detergent. A portion of the nuclei was stained with the nucleus specific dye DAPI and observed under a microscope. The treatment results showed that single, dispersed and intact single nuclei were dissociated from the frozen heart tissue samples of the mice, and the single nuclei samples after DAPI staining were visibly blue under a fluorescence microscope (fig. 12A). Adding 4% paraformaldehyde fixing solution into the rest single cell suspension for fixing, centrifuging to remove supernatant after fixing, and washing with detergent for three times. The single-cell nuclear suspension samples of the frozen heart tissue of the fixed mice were permeabilized, single-stranded DNA blocked and single-cell nuclear transcriptome sequenced as described in examples 2, 3 above. Sequencing results showed that the ratio of the detected base distribution in single nuclei of mouse cryopreserved heart tissue samples was in the normal range, with a ratio of 15.0% for intergenic regions, 6.4% for coding regions, 6.6% for UTR regions, 52.7% for intronic regions and 19.2% for ribosomal regions (fig. 12B). The results of the gene comparison showed that the number of genes that could be detected in the single nuclei of the mice cryopreserved heart tissue samples was about 3000 (fig. 12C), and the number of UMI was about 8000 (fig. 12D).

Example 8: sequencing isolated single cells of fresh islet tissue of mice

Cutting fresh islet tissue of a mouse into 1-3 mm ³ Adding single cell dissociation liquid containing 0.15% collagenase I and 0.15% disperse enzyme with twice tissue volume, reacting at 37 ℃ for 30 minutes to digest islet tissue, filtering through 100 μm, 70 μm and 40 μm cell sieves respectively, and washing with detergent three times to prepare single cell suspension. The treatment results revealed that dispersed, intact single cells were dissociated from the fresh islet tissue samples of mice, but that few cells were still aggregated (fig. 13A). The single cells of the fresh islet tissue of the mice were fixed with reference to the method described in example 7 above, and the single cells of the fixed fresh islet tissue of the mice were permeabilized, single-stranded DNA blocked, and single-cell transcriptome sequenced with reference to the methods described in examples 2, 3 above. Sequencing results showed that the ratio of the detected base distribution in single cell samples of fresh islet tissue of mice not subjected to single-strand DNA blockingMost of the examples are intergenic regions with little coding region ratio (FIG. 13B), and the base distribution ratio detected in single-stranded DNA-blocked single-cell samples of fresh islet tissue of mice is in the normal range, wherein the ratio of intergenic regions is 12.4%, the ratio of coding regions is 3.5%, the ratio of UTR regions is 6.1%, the ratio of intronic regions is 59.4%, and the ratio of ribosomal regions is 18.7% (FIG. 13C). The gene alignment showed that the number of genes that could be detected in single nuclei of single cell samples of fresh islet tissue of mice was about 2000 (fig. 13D) and the number of UMI was about 5000 (fig. 13E).

Example 9: proteinase K treated fresh islet tissue single cell samples from mice

Single cells were isolated from fresh islet tissue samples of mice by the method described in example 8 above, and then 100ul proteinase K (5-15 mg/ml) was added to the filtered mixture, which was reacted at 25-50℃for 5-30 minutes, centrifuged at room temperature and washed three times with detergent to prepare a single cell suspension. The treatment results showed that single cells with cleaner background and higher single dispersion were dissociated from the fresh islet tissue samples of the mice (fig. 14A). The single cells of the fresh islet tissue of the mice were fixed with reference to the method described in example 5 above, and the single cells of the fixed fresh islet tissue of the mice were permeabilized, single-stranded DNA blocked, and single-cell transcriptome sequenced with reference to the methods described in examples 2, 3 above. Sequencing results showed that the proportion of base distribution detected in single-stranded DNA-blocked single-cell samples of fresh islet tissue of mice treated with proteinase K was in the normal range, with 15.7% of intergenic regions, 6.3% of coding regions, 7.9% of UTR regions, 41.4% of intronic regions and 28.8% of ribosomal regions (FIG. 14B). The gene comparison result shows that the number of genes which can be detected in single nuclei of a single cell sample of fresh islet tissue of a mouse is about 4000 (fig. 14C), and the number of UMIs is about 10000 (fig. 14D).

The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that while the present invention is not limited to the foregoing embodiments, the foregoing embodiments and description are merely illustrative of the principles of this invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, and these changes and modifications fall within the scope of the invention as hereinafter claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method of obtaining a single cell or single cell nuclear sample, the method comprising performing single strand DNA blocking.

2. The method of claim 1, wherein the single-stranded DNA blocking comprises an in situ extension reaction on a naked DNA single strand in the nucleus; preferably, the in situ extension reaction occurs by the addition of a DNA blocking reagent; more preferably, the DNA blocking reagent comprises a DNA elongase, a blocking primer, dntps, and a reaction buffer; optionally, the DNA blocking reagent comprises TritonX-100, PEG8000, mgCl ₂ And/or MgSO ₂ 。

3. A method as claimed in claim 1 or 2, characterized in that the method comprises the steps of:

(1) Preprocessing an original sample;

(2) Dissociation of the pretreated sample to obtain single cells or single nuclei; and

(3) Single stranded DNA blocking.

4. The method of claim 3, wherein said pre-treated sample dissociation comprises further treating said sample with proteinase K after adding single cell dissociation fluid or single cell nuclear dissociation fluid to said pre-treated sample to obtain single cells or single cell nuclei; preferably, the concentration of the proteinase K is 5-15 mg/ml; more preferably the treatment time is from 5 to 30 minutes.

5. The method of claim 3 or 4, wherein the single cell dissociation solution is an enzyme-or chelator-containing buffer; more preferably, the enzyme is selected from collagenase, neutral protease, trypsin, elastase, hyaluronidase, papain, deoxyribonuclease i; preferably, the buffer is selected from PBS, HEPES, TRIS or SSC buffers.

6. The method of claim 3 or 4, wherein the single cell nuclear dissociation solution is a buffer containing a nonionic surfactant; preferably, the nonionic surfactant is selected from NP-40, triton X-100, tween-20, tween-80, CHAPS detergent, brij-58, octyl Thioglucoside (OTG); more preferably, the buffer is selected from PBS, HEPES, TRIS or SSC buffers.

7. The method of any one of claims 3-6, comprising performing the immobilization of the single cells or nuclei by adding an immobilization fluid after step (2) and before step (3), the immobilization fluid being a simple immobilization fluid or a mixed immobilization fluid; 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 mercuric chloride; preferably, the mixed fixative solution includes, but is not limited to, acetic acid-alcohol mixed solution, formalin-acetic acid-alcohol solution, and Bao Yinshi fixative solution.

8. The method of claim 7, wherein said method is performed by adding a permeabilizing agent after the immobilization of said single cell or single cell nucleus and prior to step (3); preferably, permeabilization of the single cell nucleus is performed by adding a buffer comprising a permeabilizing agent; wherein the permeabilizing agent is preferably Triton X-100, NP-40, a saponin, tween 20 or Tween80, more preferably a digitonin; wherein the buffer is preferably PBS, HEPES, TRIS or SSC buffer.

9. The method of any one of claims 1-8, wherein the original sample is a paraffin-embedded sample, a frozen sample, a fresh sample, a frozen sample, a fixed fluid preservation sample, or a sample preservation fluid preservation sample.

10. Single cell or single cell nuclear sample obtained by treatment with the method of any one of claims 1-9.

11. Use of the method according to any one of claims 1-9 or the single cell or single cell nuclear sample according to claim 10 in single cell, single cell nuclear, single microorganism transcriptome sequencing; preferably the use is in the fields of microbiology, basic medicine, clinical medicine, agrology, cell biology, immunology, developmental biology, pathology, neurobiology and development, genetics, stem cells, tumour, reproductive health, metagenomics and microecology, and new drug development.