A method for obtaining a single cell or single nucleus sample, a sample obtained using the same and applications

Abstract

The present invention provides a method for obtaining single cell or single nucleus samples, cells or nuclei obtained using the method and their use in transcriptome sequencing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically to the field of single-cell sequencing, and more specifically to a method for obtaining single-cell or single-cell nucleus samples, the use of the obtained samples, and their applications. Background Technology

[0002] Currently, the most widely used high-throughput single-cell transcriptome sequencing technology is the droplet microfluidic platform-based single-cell sequencing technology developed by 10X Genomics. This technology can label, sequence, and analyze thousands of cells, obtaining gene expression profiles at the single-cell level, enabling cell subpopulation segmentation and detection of differentially expressed genes between cell subpopulations. Similar technologies include inDrop and Drop-seq. 10X Genomics' single-cell transcriptome sequencing technology uses polyT primers to capture RNA containing polyA tails in samples, but its detection sensitivity is low. Samples preserved through cryopreservation, refrigeration, fixation, sample preservation solutions, or FFPE are prone to DNA double-strand structure disruption, exposing single-stranded DNA. If existing single-cell transcriptome sequencing technology is directly applied to these samples, the reverse transcription primers may simultaneously capture cDNA information from the DNA, resulting in the sequencing of numerous intergenic regions, wasting sequencing bases, and contaminating the gene expression matrix. Summary of the Invention

[0003] To address the aforementioned problems, in a first aspect, the object of the present invention is to provide a method for obtaining a single-cell or single-cell nucleus sample, the method comprising blocking single-stranded DNA.

[0004] In a specific implementation, the single-stranded DNA blocking includes an in situ extension reaction on the naked DNA single strand in the cell nucleus.

[0005] In specific embodiments, the method of the present invention involves adding a DNA blocking reaction reagent to induce the in situ extension reaction. In a preferred embodiment, the DNA blocking reaction reagent comprises DNA elongase, blocking primers, dNTPs, and a reaction buffer. In an optional embodiment, the DNA blocking reaction reagent may further comprise Triton X-100, PEG8000, MgCl2, and / or MgSO2.

[0006] In a specific implementation, the method of the present invention includes the following steps: (1) pretreatment of the original sample; (2) dissociation of the pretreated sample to obtain a single cell or a single cell nucleus; and (3) blocking of single-stranded DNA.

[0007] In a specific implementation, the pretreated sample dissociation includes adding a single-cell dissociation solution or a single-nucleus dissociation solution to the pretreated sample to dissociate the sample, followed by further treatment with proteinase K to obtain single cells or single nuclei. In a preferred embodiment, the concentration of proteinase K is 5–15 mg / ml. In a more preferred embodiment, the treatment time is 5–30 minutes.

[0008] In a specific embodiment, the single-cell dissociation solution is a buffer solution containing an enzyme or a chelating agent; wherein the enzyme may be selected from, but is not limited to, collagenase, neutral protease, trypsin, elastase, hyaluronidase, papain, and deoxyribonuclease I; the buffer solution may be selected from, but is not limited to, PBS, HEPES, TRIS, or SSC buffer. In a preferred embodiment, the concentration of the enzyme in the single-cell dissociation solution is 0.01% to 0.5% g / 100 ml. In another preferred embodiment, the chelating agent is EDTA; the concentration of the chelating agent in the single-cell dissociation solution is 0.001% to 0.1% g / 100 ml.

[0009] In a specific implementation, the single-cell nucleus dissociation solution is a buffer solution containing a nonionic surfactant, wherein the concentration of the nonionic surfactant can be 0.05-0.5% (volume ratio), and the nonionic surfactant can be selected from, but is not limited to, NP-40, Triton X-100, Tween-20, Tween-80, CHAPS detergent, Brij-58, and octyl glucosinolate (OTG); the buffer solution can be selected from, but is not limited to, PBS, HEPES, TRIS, or SSC buffer.

[0010] In a specific implementation, the single-cell nucleus dissociation of the method of the present invention includes adding a nonionic surfactant to a pretreated sample and then homogenizing, shaking or ultrasonically breaking it to form a single-cell nucleus suspension.

[0011] In specific embodiments, the method of the present invention may include fixing the single cell or single cell nucleus by adding a fixative after step (2) and before step (3), wherein the single cell or single cell nucleus is fixed by adding a fixative in step (3), and the fixative may be a simple fixative or a mixed fixative. In a preferred embodiment, the simple fixative may be selected from, but is not limited to, paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, and mercuric chloride. In another preferred embodiment, the mixed fixative may be selected from, but is not limited to, an acetic acid-alcohol mixture, a formalin-acetic acid-alcohol solution, and Boeinger's fixative. In specific embodiments, the fixation time may be selected according to the sample being processed, for example, 15 min to 30 min or overnight.

[0012] In specific embodiments, the method of the present invention may include permeabilizing the single cell or single cell nucleus by adding a permeabilizing agent after fixation and before step (3). In a preferred embodiment, the single cell nucleus may be permeabilized by adding a buffer containing the permeabilizing agent. In a preferred embodiment, the permeabilizing agent may be selected from, but is not limited to, Triton X-100, NP-40, saponins, Tween 20, and Tween 80, wherein the saponins are preferably digitalis saponins. In a more preferred embodiment, the content of the permeabilizing agent is 0.1-0.5% (volume ratio); in another preferred embodiment, the buffer may be selected from, but is not limited to, PBS, HEPES, TRIS, and SSC buffer.

[0013] In specific implementation schemes, the original sample may be, but is not limited to, paraffin-embedded samples, cryopreserved samples, refrigerated samples, fresh samples, samples preserved in fixative or sample preservation solutions.

[0014] In a second aspect, the object of the present invention is to provide single-cell or single-cell nucleus samples obtained by the method of the first aspect.

[0015] In a third aspect, the object of the present invention is to provide the application of the method in the first aspect or the single-cell or single-nucleus sample obtained by the method in the first aspect in transcriptome sequencing of single cells, single nuclei, or single microorganisms; preferably, the application is in the fields of microbiology, basic medicine, clinical medicine, agronomy, cell biology, immunology, developmental biology, pathology, neurobiology and development, genetics, stem cells, oncology, reproductive health, metagenomics and microecology, and new drug development.

[0016] In specific implementations, the transcriptome sequencing technology in the method of this invention can be a variety of single-cell or single-nucleus transcriptome sequencing technologies based on different principles. These single-cell or single-nucleus transcriptome sequencing technologies include, but are not limited to, the 10X Genomics platform, the BD Rhapsody platform, VASA-seq, Drop-seq, Smart-seq, Split-pool, or single-cell or single-nucleus transcriptome sequencing technologies developed by this laboratory (e.g., those described in Chinese Patent Application No. 202210174619.9).

[0017] The method of this invention produces cleaner single-cell or single-nucleus samples with significantly reduced residual debris on the surface of single cells or nuclei. When used for single-cell or single-nucleus transcriptome sequencing, the proportion of bases aligned to intergenic regions is significantly reduced, while the proportion aligned to coding regions, UTR regions, intron regions, and ribosomal regions is significantly increased. This significantly improves the utilization rate of the sequencing bases and reduces contamination of gene expression levels by intergenic regions. Therefore, compared with existing technologies, the method of this invention enables higher sensitivity, lower cost, and more accurate gene expression matrices for single-cell or single-nucleus transcriptome sequencing. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the method for obtaining single-cell or single-cell nucleus samples according to the present invention.

[0019] Figure 2 This is a schematic diagram illustrating the principle of single-cell or single-nucleus transcriptome sequencing using the method of this invention.

[0020] Figure 3 These are single-cell micrographs of mouse FFPE kidney samples after processing with the method of this invention.

[0021] Figure 4 This is a base distribution map of single-cell sequencing data from mouse FFPE kidney samples after treatment with no single-stranded DNA blocking, treatment with single-stranded DNA blocking, and treatment with DNase digestion.

[0022] Figure 5 These are microscopic images of single-cell nucleus samples from mouse FFPE kidneys treated with proteinase K and collagenase.

[0023] Figure 6 These are single-cell DAPI staining images of various mouse FFPE tissue samples processed by the method of this invention.

[0024] Figure 7 This is a base distribution map of single cell nuclei from various mouse FFPE tissue samples processed by the method of this invention.

[0025] Figure 8 This refers to the number of single-cell nuclear genes detected in various mouse FFPE tissue samples processed by the method of this invention.

[0026] Figure 9 This is the analysis result of single-cell nuclear sequencing data of various mouse FFPE tissue samples processed by the method of this invention.

[0027] Figure 10 These are the results of single-cell nucleus isolation and sequencing data analysis of clinical human liver cancer FFPE samples processed by the method of this invention.

[0028] Figure 11 These are differentially expressed genes from cell populations in single-cell nuclear sequencing data of clinical human liver cancer FFPE samples processed by the method of this invention.

[0029] Figure 12 These are the results of single-cell nucleus isolation and sequencing data analysis of mouse cryopreserved heart tissue samples processed by the method of this invention.

[0030] Figure 13 These are the results of single-cell isolation and sequencing data analysis of fresh mouse pancreatic islet muscle tissue samples processed by the method of this invention.

[0031] Figure 14 The results are from the treatment of fresh mouse islet tissue single-cell samples with proteinase K and the analysis of sequencing data. Invention Details

[0033] An embodiment of the present invention provides a method for obtaining single-cell or single-cell nucleus samples, which avoids hybridization and extension reactions between the blocking primers added during reverse transcription and the exposed single-stranded DNA in the sample due to the disruption of the double-stranded structure by blocking the DNA. Figure 1 As shown, the method mainly includes pretreatment of the original sample, obtaining single cells or single cell nuclei from the pretreated sample, and blocking single-stranded DNA. Optionally, the single cells or single cell nuclei are fixed and permeabilized before blocking the single-stranded DNA. Specifically, the method includes the following steps: selecting the region of interest for FFPE samples, frozen samples, refrigerated samples, fixed samples, or samples preserved in sample preservation solution; taking 5-50 mg of sample from this region using a punch or cutting thin slices larger than 50 μm using a slide, and performing corresponding pretreatment; adding a single-cell or single-cell nucleus dissociation solution to dissociate and obtain single cells or single cell nuclei; optionally, after dissociating the sample with the single-cell or single-cell nucleus dissociation solution, further processing the sample with proteinase K to obtain cleaner single cells or single cell nuclei; optionally, fixing and permeabilizing the obtained single cells or single cell nuclei; and performing an in-situ extension reaction on the nucleus of the single cell or the naked single-stranded DNA in the single cell nucleus to block the naked single-stranded DNA.

[0034] definition

[0035] The single cells mentioned in this article include, but are not limited to, eukaryotic single cells, prokaryotes (bacteria, actinomycetes, rickettsiae, chlamydiae, mycoplasma, cyanobacteria, and archaea, etc.), single-celled algae, viruses, etc., and the single cell nuclei mentioned in this article include, but are not limited to, the nuclei of the above-mentioned cells.

[0036] In this article, cryopreserved samples refer to human or animal / plant tissue or cell samples frozen at 0℃ to -80℃ or in liquid nitrogen.

[0037] In this article, refrigerated samples refer to human, animal, or plant tissue or cell samples stored at 0°C to 8°C.

[0038] In this article, "fixed samples" refers to human or animal / plant tissue or cell samples preserved in paraformaldehyde or formaldehyde.

[0039] The samples preserved in this article refer to those preserved in RNAwait (a non-frozen tissue RNA preservation solution). Tissue preservation solutions and other commercially available sample preservation solutions contain human, animal, or plant tissue or cell samples.

[0040] In this article, FFPE samples refer to paraffin sections or blocks formed by fixing human or animal tissue or cell samples with formalin and embedding them in paraffin.

[0041] The RNAs discussed in this article include detectable coding RNAs and various forms of non-coding RNAs, such as miRNAs, lncRNAs, siRNAs, and circRNAs.

[0042] The different types of cells mentioned in this article can be cells from different species or cells from the same species (e.g., cells from different culture batches or cells from different sources).

[0043] The blocking primers of this invention contain 3 to 20 random base sequences (i.e., 5'-(NNN)). 3-20 Composed of dG, dA, dT or dC, it can randomly bind to single-stranded DNA sequences.

[0044] The detergents used in this article can be PBS, HEPES, TRIS or SSC-based buffers. 0.005 to 0.2% of nonionic surfactants, such as Tween 20, Tween 80, Triton X-100, etc., can be added to the detergents.

[0045] The main steps of the sequencing method used in the embodiments of the present invention will be described in general below.

[0046] Sample preprocessing

[0047] Appropriate sample pretreatment methods can be selected for different types of samples to prepare samples of appropriate size that can be dissociated into single-cell suspensions and do not contain reagents that affect subsequent permeation, blocking and other reactions.

[0048] First, samples of appropriate size can be collected using methods such as scissors, blade cutting, tissue slicers, punching, laser capture microdissection (LMD or LCM), and microarea sampling.

[0049] Then, appropriate pretreatment methods can be selected according to different types of samples. For paraffin embedding...

[0050] For (FFPE) samples, use a dewaxing agent to remove paraffin and add ethanol of varying concentrations for rehydration; for frozen samples, thaw quickly and wash three times with detergent; for refrigerated samples, wash three times with detergent; for samples preserved in fixative, remove the fixative and wash three times with detergent; for samples preserved in sample preservation solution, remove the preservation solution and wash three times with detergent.

[0051] The dewaxing agent for removing paraffin from FFPE samples can be selected from xylene, dextrorotatory limonene, water-based dewaxing agents, environmentally friendly transparent agents, Histo-Clear tissue dewaxing agent, Histo-Clear II environmentally friendly tissue transparent agent, and Seebio non-toxic environmentally friendly transparent dewaxing agent. The ethanol concentration gradient for rehydration can be 100%, 98%, 95%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, etc.

[0052] Preparation of single-cell nucleus suspension

[0053] Appropriate single-nucleus dissociation methods can be selected for different types of samples to prepare single-nucleus suspensions. The aim is to obtain a large number of single, dispersed single nuclei with intact nuclei morphology. Single-nucleus dissociation solution is added to the pretreated sample for dissociation. To improve the dissociation effect, the concentration of nonionic surfactant in the dissociation solution can be adjusted, or multiple different nonionic surfactants can be used. Alternatively, the sample with the dissociation solution can be homogenized using a homogenizer, shaken on a shaker, or sonicated using an ultrasonic homogenizer. Proteinase K can then be added to the sample to obtain even cleaner single nuclei. After treatment, the supernatant is removed by centrifugation to obtain single nuclei. A portion of the nuclei is stained and observed under a microscope.

[0054] Preparation of single-cell suspension

[0055] Appropriate single-cell dissociation methods can be selected for different types of samples to prepare single-cell suspensions, aiming to obtain a large number of individual, dispersed single cells with intact cell morphology. Single-cell dissociation solution is added to the pretreated sample, and the digestion reaction is carried out by incubation at 37°C. Proteinase K can then be added to obtain even cleaner single cells. After treatment, the sample is filtered and washed to prepare a single-cell suspension.

[0056] Single-cell or single-nucleus fixation

[0057] Appropriate fixation reagents and fixation times can be selected for different types of samples to crosslink and fix substances such as RNA, DNA and proteins in single cells or single cell nuclei into single cells or single cell nuclei, so that single cells or single cell nuclei can maintain the complete cell morphology and internal structure in subsequent permeabilization, transcriptome sequencing and other reaction steps.

[0058] After preparing the sample into a single-cell nucleus suspension, fixative is added for fixation. After fixation, the supernatant is removed by centrifugation, and the sample is washed three times with detergent. During the procedure, a suitable fixative can be selected based on the characteristics of different sample types. Fixatives include, but are not limited to, simple fixatives such as paraformaldehyde, formaldehyde, formalin, methanol, acetone, ethanol, acetic acid, picric acid, chromic acid, potassium dichromate, and mercuric chloride, as well as mixed fixatives such as acetic acid-alcohol mixtures, formalin-acetic acid-alcohol solutions, and Boeinger's fixative. Different fixation times can be selected according to specific circumstances, such as 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours. Different fixation temperatures can be selected according to specific circumstances, such as 4°C, on ice, or room temperature. For samples preserved with fixative or FFPE samples, the fixation step can be omitted or the fixation time reduced.

[0059] The method for fixing single cells is basically the same as that for fixing single cell nuclei.

[0060] Single-cell or single-cell nucleus permeation

[0061] Appropriate permeation reagents and permeation times can be selected for different types of samples to create pores in the cell membrane or nuclear membrane of a single cell, allowing subsequent reaction reagents to better enter and exit the cell or nucleus. The permeation method for single cells is basically the same as that for single nuclei.

[0062] After fixation, add a permeabilizing agent to the sample and permeabilize. After permeabilization, centrifuge to remove the supernatant and wash three times with detergent. During the procedure, select a suitable permeabilizing agent based on the characteristics of different sample types. Suitable permeabilizing agents include 0.1–0.5% (v / v) Triton X-100, NP-40, saponins, Tween 20, Tween 80, digitalis saponins, or buffers containing 100 μM Digitonin or 0.5% Saponin. Buffers can be PBS, HEPES, TRIS, or SSC buffers. The permeabilization time can be selected from 1 to 15 minutes depending on the characteristics of different sample types.

[0063] Single-stranded DNA blocking

[0064] The single-stranded DNA blocking reaction involves an in-situ extension reaction of the naked single-stranded DNA using blocking primers, thereby blocking the naked single-stranded DNA into double-stranded DNA, preventing it from participating in subsequent reverse transcription reactions, and ultimately obtaining single-cell or single-nucleus samples that can be used for subsequent single-cell or single-nucleus transcriptome sequencing.

[0065] DNA blocking reagents, including DNA elongase, blocking primers, dNTPs, and reaction buffer, are added to permeabilized single-cell or single-cell nucleus samples. The mixture is reacted at 37°C for 10–90 minutes. After the reaction, the sample is washed three times with detergent. To improve the blocking effect, Triton X-100, PEG8000, MgCl2, or MgSO2 can be added to the DNA blocking reagent.

[0066] Single-cell or single-cell nuclear transcriptome sequencing

[0067] After permeabilization, various types of single-cell or single-nucleus transcriptome sequencing technologies can be used for sequencing. These technologies include, but are not limited to, the 10X Genomics platform, VASA-seq, Drop-seq, Smart-seq, Split-pool, or transcriptome sequencing technologies developed by the inventors of this application (e.g., those described in Chinese Patent Application No. 202210174619.9) (see [link to patent application]). Figure 2 ). Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0069] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0071] Example 1: Dissociation of single-cell nuclei from mouse FFPE kidney samples

[0072] Under a microscope, select the region of interest on a mouse FFPE kidney sample. Use a punch to remove approximately 10 mg of sample from the paraffin block and place it in a 1.5 ml centrifuge tube. Add 1 ml of xylene and incubate at room temperature for 5–10 minutes. Remove the xylene and add a fresh 1 ml of xylene for dewaxing. After dewaxing, remove the xylene and rehydrate by adding ethanol at gradient concentrations of 100%, 95%, 80%, 70%, 50%, and 30%, respectively. After rehydration, 1 ml of single-cell nucleus lysis buffer was added. The single-cell nucleus lysis buffer was 2X SSC buffer containing 0.5–2% NP-20 ionic surfactant and MgCl2 (1–5 mM). The sample and lysis buffer mixture was added to a Durns homogenizer and homogenized 10–30 times. The mixture was then incubated on ice for 5–15 minutes. A portion of the homogenized mixture was taken and 100 μL of proteinase K (5–15 mg / ml) was added. The mixture was incubated at 25–50 °C for 5–30 minutes, centrifuged at room temperature, and the supernatant was removed to obtain single cell nuclei. The nuclei were washed three times with washing buffer. A portion of the cell nuclei were stained with the nuclear-specific dye DAPI and observed under a microscope. See the results below. Figure 3 As can be seen, some scattered and intact FFPE single cell nuclei can be dissociated from mouse FFPE kidney samples lysed with single cell nucleus lysis buffer. The single cell nuclei samples stained with DAPI appear distinctly blue under a fluorescence microscope, but tissue fragments still remain. Figure 3 A), after filtration through a 40μm cell sieve, a relatively clean and dispersed FFPE single-cell nucleus suspension can be obtained, which can be used for the next step of the experiment. Figure 3 B).

[0073] Example 2: Single-cell nucleus permeation and single-stranded DNA blocking

[0074] The mouse FFPE kidney single-cell nucleus samples obtained in Example 1 were permeabilized, single-stranded DNA blocked, and single-cell nucleus whole transcriptome sequenced. The specific methods are as follows:

[0075] Permeability

[0076] Take an appropriate amount of the single-cell nucleus sample treated in Example 1, add a permeabilizing agent containing 0.2% Triton X-100 and permeabilize for 10 minutes. After permeabilization, centrifuge to remove the supernatant and wash three times with detergent.

[0077] Single-stranded DNA blocking

[0078] Add DNA blocking reagent containing DNA elongase (1-10 U / μl), blocking primer (5-50 mM), dNTPs (1-10 mM), and 1X ThermoPol reaction buffer to the permeabilized sample. React at 37°C for 20-45 minutes. After blocking, wash three times with detergent.

[0079] Example 3: Sequencing of mouse FFPE kidney samples

[0080] Reverse transcription

[0081] Obtained in Example 2 Single-stranded DNA blocking Reverse transcription was performed on mouse FFPE kidney single-cell nucleus samples by adding reverse transcriptase, reverse transcription buffer, dNTPs, and reverse transcription primers. After the reaction, the samples were washed three times with detergent.

[0082] Add capture connector

[0083] Terminal transferase, dCTP, and reaction buffer were added to the reverse transcribed sample, and the mixture was incubated at 37°C for 30 minutes. After the reaction, the sample was washed three times with detergent.

[0084] Cellular septum

[0085] Single-cell nucleus samples, extension reaction reagents (including DNA polymerase, dNTPs, and reaction buffer), coded microspheres, and oil phase are added to syringes, which are then connected to the corresponding inlets of the microfluidic chip via tubing. An appropriate flow rate is set to form a water-in-oil droplet containing a single single-cell nucleus, a single coded microsphere, and extension reaction reagents. The droplets are collected to form a single-chamber cell separator containing a single cell.

[0086] The second strand of cDNA synthesis

[0087] The collected single droplets were aliquoted into different tubes and subjected to an extension reaction to synthesize a second strand of barcoded cDNA within the single droplet. After the extension reaction, the single droplets were broken up, and the cDNA in the extraction tube was purified using magnetic beads.

[0088] Library construction and high-throughput sequencing

[0089] A portion of double-stranded cDNA was used as a template for qPCR to detect the total cDNA content. The remaining cDNA was amplified by PCR, and the amplified cDNA underwent end repair and A-tailing using the TA cloning adapter ligation library construction method. Adapters were then added using a library construction kit. The constructed library was then sequenced using the Illumina sequencing platform for high-throughput sequencing.

[0090] Sequencing results showed that the proportion of intergenic regions detected in single-cell nuclei samples from mouse FFPE kidneys after single-stranded DNA blocking was significantly reduced, from 61.2% to 11.3%; correspondingly, the proportions of coding regions, UTR regions, intron regions, and ribosomal regions all significantly increased. The proportion of coding regions increased from 1.1% to 4.0%, the proportion of UTR regions from 1.2% to 6.9%, the proportion of intron regions from 36.5% to 68.7%, and the proportion of ribosomal regions from 0.0% to 9.1%. Figure 4 A, B).

[0091] To eliminate contamination of experimental results by single-stranded DNA, we also explored the method of digesting DNA from single cells or single cell nuclei using DNase. Mouse FFPE kidney single-cell nucleus samples obtained according to the method described in Example 1 were permeabilized according to the permeabilization method described in Example 2. DNase and reaction buffer were added to the permeabilized mouse FFPE kidney single-cell nucleus samples, and the mixture was incubated at 37°C for 30–60 minutes. 1 μL of 25 mM EDTA was added to the reaction system, and the mixture was incubated at 65°C for 10 minutes to inactivate the DNase. After the reaction, the samples were washed three times with detergent. The method described in Example 3 above was used to reverse transcribe, add capture adapters, separate cells, synthesize the second strand of cDNA, construct libraries, and perform high-throughput sequencing on the DNA-digested mouse FFPE kidney single-cell nucleus samples. The results showed that the proportions of intergenic regions, coding regions, UTR regions, intronic regions, and ribosomal regions detected by sequencing in the DNA-digested mouse FFPE kidney single-cell nucleus samples did not change significantly. Figure 4 C) indicates that the use of DNase digestion cannot effectively eliminate the contamination of experimental results by single-stranded DNA.

[0092] Example 4: Proteinase K treatment of mouse FFPE kidney single-cell nucleus samples

[0093] Following the method described in Example 1 above, mouse FFPE kidney tissue samples underwent sample pretreatment and single-cell nucleus dissociation. Then, 100 μL of proteinase K (5–15 mg / mL) was added to a portion of the homogenized mixture, and the mixture was reacted at 25–50 °C for 5–30 minutes. After centrifugation at room temperature, the supernatant was removed to obtain single cell nuclei, which were then washed three times with washing buffer. A portion of the cell nuclei were stained with the nuclear-specific dye DAPI and observed under a microscope. The processing results are described in [link to relevant documentation]. Figure 5 As can be seen, the background of single-cell nucleus samples from mouse FFPE kidneys treated with proteinase K is cleaner, the nuclei are all single and dispersed, and they maintain an intact nucleus morphology. Figure 5 A).

[0094] To compare the effects with the treatment using proteinase K described above, a control experiment was also conducted using collagenase to treat single cell nuclei. A portion of the homogenized mixture was taken, and 100 μL of collagenase (5–15 mg / mL) was added. The mixture was reacted at 25–50 °C for 5–30 minutes, centrifuged at room temperature, and the supernatant was removed to obtain single cell nuclei. The nuclei were washed three times with washing buffer. A portion of the nuclei were stained with the nuclear-specific dye DAPI and observed under a microscope. The results are shown in [link to results]. Figure 5 B, it can be seen that the surface of the single cell nucleus treated with collagenase has more cytoplasm, and some cells are still stuck together. Figure 5 B), therefore, proteinase K is more effective than other proteases in dissociating single-cell nuclei.

[0095] Example 5: Sequencing of various FFPE tissue samples from mice

[0096] Following the methods described in Examples 1-4 above, various mouse FFPE tissue samples (including liver, kidney, heart, and testis) underwent sample pretreatment, single-cell nucleus dissociation, proteinase K treatment, permeabilization, single-stranded DNA blocking, reverse transcription, addition of capture adapters, cell septation, synthesis of the second strand of cDNA, library construction, and high-throughput sequencing. The results showed that intact cell nuclei could be isolated from various mouse FFPE tissue samples. Figure 6 (AD). Sequencing results showed that the base distribution ratios detected in single cell nuclei of various FFPE tissue samples from mice were all within the normal range, with the proportions of intergenic regions ranging from 11.9% to 29.8%, coding regions from 4.1% to 7.6%, UTR regions from 5.4% to 6.7%, intronic regions from 31.7% to 53.8%, and ribosome regions from 18.1% to 28.9%. Figure 7 AD). Gene alignment results showed that the number of genes detectable in single cell nuclei of various FFPE tissue samples from mice was 3000–4000 (AD). Figure 8 Cluster analysis of the calculated single-cell nuclear transcriptome expression matrix showed that these cells from various FFPE tissue samples from mice could be clustered into multiple cell populations. Figure 9 A), and by examining the expression of reported marker genes in these cell populations ( Figure 9 B) can identify the cell types of these cell populations. Figure 9 A).

[0097] Example 6: Sequencing of clinical FFPE human liver cancer tissue samples

[0098] Following the methods described in Examples 1-4 above, clinical human hepatocellular carcinoma FFPE samples underwent sample preprocessing, single-nucleus dissociation, proteinase K treatment, permeabilization, single-stranded DNA blocking, and single-nucleus transcriptome sequencing. The results showed that intact cell nuclei could be isolated from the clinical human hepatocellular carcinoma FFPE samples. Figure 10 A). Sequencing results showed that the base distribution ratios detected in single cell nuclei of clinical human hepatocellular carcinoma FFPE samples were all within the normal range, with the proportions of intergenic regions being 8.0%, coding regions 12.0%, UTR regions 19.0%, intron regions 45.8%, and ribosomal regions 15.3%. Figure 10 B). Gene alignment results showed that approximately 3000 genes could be detected in a single cell nucleus of clinical human hepatocellular carcinoma FFPE samples. Figure 10 C), the UMI number is around 10,000. Figure 10 D). Cluster analysis of the calculated single-cell nuclear transcriptome expression matrix showed that these cells from clinical human hepatocellular carcinoma FFPE samples could be clustered into multiple cell populations, and the cell types of these cell populations could be identified based on the expression of reported marker genes in these cell populations, including most cell types in human liver tissue (hepatocyte 1, hepatocyte 2, hepatocyte 3, hepatic macrophages, hepatic satellite cells, T cells, B cells). Figure 10 E). By calculating differentially expressed genes among different cell populations, new marker genes can be identified. Figure 11 ).

[0099] Example 7: Sequencing of frozen mouse heart tissue samples

[0100] Rapidly thaw frozen mouse heart tissue samples. Under a microscope, select the region of interest on the frozen mouse heart tissue sample, and use a punch to extract approximately 10 mg of sample into a centrifuge tube. Add pre-chilled detergent and wash three times. Add 1 ml of single-cell nucleus lysis buffer (containing 0.5–2% NP-20 nonionic surfactant and 1–5 mM MgCl2 in 2X SSC buffer). Add the sample and lysis buffer mixture to a Durns homogenizer and homogenize 10–30 times. Incubate on ice for 5–15 minutes. Add 100 μL of proteinase K (5–15 mg / ml) to the homogenized mixture and react at 25–50 °C for 5–30 minutes. Centrifuge at room temperature, remove the supernatant to obtain single cell nuclei, and wash three times with detergent. Stain a portion of the cell nuclei with the nuclear-specific dye DAPI and observe under a microscope. The results showed that single, intact, dispersed nuclei could be isolated from frozen mouse heart tissue samples. The single-nucleus samples stained with DAPI appeared distinctly blue under a fluorescence microscope. Figure 12 A). The remaining single-cell suspension was fixed with 4% paraformaldehyde fixative. After fixation, the supernatant was removed by centrifugation, and the cells were washed three times with detergent. The fixed mouse cryopreserved heart tissue single-cell nucleus suspension samples were permeabilized, single-stranded DNA blocked, and single-cell nuclear transcriptome sequenced according to the methods described in Examples 2 and 3 above. Sequencing results showed that the base distribution ratios detected in the single-cell nuclei of the mouse cryopreserved heart tissue samples were all within the normal range, with the proportion of intergenic regions at 15.0%, coding regions at 6.4%, UTR regions at 6.6%, intron regions at 52.7%, and ribosome regions at 19.2%. Figure 12 B). Gene alignment results showed that approximately 3000 genes could be detected in the nuclei of single cells in frozen mouse heart tissue samples. Figure 12 C), the UMI number is around 8000 ( Figure 12 D).

[0101] Example 8: Sequencing of single cells isolated from fresh mouse islet tissue

[0102] Fresh mouse islet tissue was cut into 1-3 mm pieces. 3 Small pieces of pancreatic islet tissue were added to a single-cell dissociation solution containing 0.15% collagenase I and 0.15% dispersant enzyme at twice the tissue volume. The mixture was incubated at 37°C for 30 minutes to digest the islet tissue. After digestion, the tissue was filtered through 100μm, 70μm, and 40μm cell sieves, and washed three times with detergent to prepare a single-cell suspension. The results showed that dispersed and intact single cells could be dissociated from fresh mouse islet tissue samples, but a few cell clusters remained. Figure 13A). Single cells from fresh mouse islet tissue were fixed according to the method described in Example 7 above. The fixed single cells were then permeabilized, had their single-stranded DNA blocked, and underwent single-cell transcriptome sequencing according to the methods described in Examples 2 and 3 above. Sequencing results showed that in single-cell samples from fresh mouse islet tissue that had not undergone single-stranded DNA blocking, the majority of the bases detected were in intergenic regions, with very few in coding regions. Figure 13 B), while the base distribution ratios detected in single-cell samples of fresh mouse islet tissue after single-stranded DNA blocking were all within the normal range, with the proportions of intergenic regions being 12.4%, coding regions 3.5%, UTR regions 6.1%, intron regions 59.4%, and ribosomal regions 18.7%. Figure 13 C). Gene alignment results showed that approximately 2000 genes could be detected in a single cell nucleus of a fresh mouse islet tissue single-cell sample. Figure 13 D), the UMI number is around 5000 ( Figure 13 E).

[0103] Example 9: Treatment of fresh mouse islet tissue single-cell samples with proteinase K

[0104] Single cells were isolated from fresh mouse islet tissue samples using the method described in Example 8 above. Then, 100 μL of proteinase K (5–15 mg / mL) was added to the filtered mixture, and the mixture was reacted at 25–50°C for 5–30 minutes. After centrifugation at room temperature, the cells were washed three times with detergent to prepare a single-cell suspension. The results showed that single cells with a cleaner background and higher degree of individual dispersion could be isolated from the fresh mouse islet tissue samples. Figure 14 A). Single cells from fresh mouse islet tissue were fixed according to the method described in Example 5 above. The fixed single cells were then permeabilized, had their single-stranded DNA blocked, and underwent single-cell transcriptome sequencing according to the methods described in Examples 2 and 3 above. Sequencing results showed that the base distribution ratios detected in the single-cell samples of fresh mouse islet tissue treated with proteinase K and blocked with single-stranded DNA were all within the normal range. Specifically, the proportion of intergenic regions was 15.7%, coding regions 6.3%, UTR regions 7.9%, intron regions 41.4%, and ribosomal regions 28.8%. Figure 14 B). Gene alignment results showed that approximately 4000 genes could be detected in a single cell nucleus of a fresh mouse islet tissue single-cell sample. Figure 14 C), the UMI number is around 10,000. Figure 14 D).

[0105] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for obtaining single-cell or single-cell nucleus samples, characterized in that, The method includes the following steps: (1) Preprocessing of raw samples; (2) The pretreated sample is dissociated to obtain single cells or single cell nuclei; and (3) Blocking of single-stranded DNA; The single-stranded DNA blocking comprises an in situ extension reaction on naked single-stranded DNA in the cell nucleus; wherein the in situ extension reaction is carried out by adding a DNA blocking reaction reagent; the DNA blocking reaction reagent comprises DNA elongase, blocking primers, dNTPs, and a reaction buffer; the blocking primers comprise a 3-20 random base sequence 5'-(NNN). 3-20 -3' is composed of N = dG, dA, dT or dC; The pretreated sample dissociation includes adding proteinase K to the pretreated sample to further treat the sample after dissociating the sample by adding a single-cell dissociation solution or a single-cell nucleus dissociation solution to obtain single cells or single-cell nuclei.

2. The method as described in claim 1, characterized in that, The concentration of proteinase K is 5-15 mg / ml.

3. The method as described in claim 1 or 2, characterized in that, The processing time is 5 to 30 minutes.

4. The method as described in claim 1, characterized in that, The DNA blocking reaction reagent also contains Triton X-100, PEG8000, MgCl2 and / or MgSO2.

5. The method as described in claim 1, characterized in that, The single-cell dissociation solution is a buffer solution containing enzymes or chelating agents.

6. The method as described in claim 5, characterized in that, The enzymes are selected from collagenase, neutral protease, trypsin, elastase, hyaluronidase, papain, and deoxyribonuclease I.

7. The method as described in claim 5, characterized in that, The buffer solution is selected from PBS, HEPES, TRIS, or SSC buffer.

8. The method as described in claim 1, characterized in that, The single-cell nucleus dissociation solution is a buffer solution containing a nonionic surfactant.

9. The method as described in claim 8, characterized in that, The nonionic surfactant is selected from NP-40, Triton X-100, Tween-20, Tween-80, CHAPS detergent, Brij-58, and octyl glucosinolate (OTG).

10. The method as described in claim 8, characterized in that, The buffer solution is selected from PBS, HEPES, TRIS, or SSC buffer.

11. The method as described in claim 1, characterized in that, The method includes fixing the single cell or single cell nucleus by adding a fixative solution after step (2) and before step (3), wherein the fixative solution is a simple fixative solution or a mixed fixative solution.

12. The method as described in claim 11, characterized in that, 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.

13. The method as described in claim 11, characterized in that, The mixed fixatives include, but are not limited to, acetic acid-alcohol mixtures, formalin-acetic acid-alcohol solutions, and Boein's fixative.

14. The method as described in claim 11, characterized in that, The method permeates the single cell or single cell nucleus by adding a permeabilizing agent after the fixation of the single cell or single cell nucleus and before step (3).

15. The method as described in claim 14, characterized in that, The single cell nucleus was permeabilized by adding a buffer solution containing a permeabilizing agent.

16. The method as described in claim 15, characterized in that, The permeasurant is Triton X-100, NP-40, saponin, Tween 20, or Tween 80.

17. The method as described in claim 16, characterized in that, The saponin mentioned is digitalis saponin.

18. The method as described in claim 15, characterized in that, The buffer solution is PBS, HEPES, TRIS, or SSC buffer.

19. The method as described in claim 1, 11, or 14, characterized in that, The original sample mentioned therein is a paraffin-embedded sample, a cryopreserved sample, a fresh sample, a refrigerated sample, a sample preserved in a fixative solution, or a sample preservation solution.

20. A single-cell or single-cell nucleus sample obtained by the method described in any one of claims 1-19.

21. The application of the method according to any one of claims 1-19 in transcriptome sequencing of single cells, single cell nuclei, or single microorganisms; wherein the application is not for diagnostic or therapeutic purposes.

22. The application as described in claim 21, characterized in that, The applications are in the fields of basic medicine, clinical medicine, agronomy, and new drug development.

23. The application of the single-cell or single-nucleus sample as described in claim 20 in transcriptome sequencing of single cells, single nuclei, or single microorganisms; wherein the application is not for diagnostic or therapeutic purposes.

24. The application as described in claim 23, characterized in that, The applications are in the fields of basic medicine, clinical medicine, agronomy, and new drug development.

Images