Sample treatment method for detecting accessibility of single cell chromatin and application of sample treatment method

Abstract

The invention relates to a sample treatment method for detecting accessibility of single cell chromatin and application of the sample treatment method. The sample treatment method comprises the following steps: slicing a tissue sample to obtain a tissue slice, and sequentially carrying out permeabilization treatment, transposase treatment and cell nucleus extraction treatment on the tissue slice, the tissue sample includes a frozen tissue sample. According to the method, a'slice in-situ transposition reaction 'strategy is designed, the original spatial conformation and physiological state of chromatin in vivo are effectively maintained, chromatin structure disturbance caused by early release of cell nucleuses in the thawing process is avoided, the quality of ATAC-seq data is remarkably improved, and the method is suitable for being used in a large scale. The enrichment fraction of the transcription start site and the proportion result of fragments overlapped with peaks are remarkably improved, a nucleosome positioning signal is clearer, and the accuracy and reliability of chromatin openness detection are improved.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a sample processing method for detecting the accessibility of chromatin in single cells and its application. Background Technology

[0002] Assay for Transposase-Accessible Chromatinusing Sequencing (ATAC-seq) is a high-throughput sequencing method widely used in epigenomics research to detect open regions of chromatin in the genome. The core principle of this technology is the use of a highly active mutant Tn5 transposase, which specifically recognizes and inserts into open regions of chromatin (i.e., DNA regions sparsely or absently occupied by nucleosomes), simultaneously fragmenting sequencing adapters and ligating them to both ends of the target DNA during insertion. Subsequently, PCR amplification and high-throughput sequencing yield a map of chromatin accessibility across the entire genome. If a sample contains multiple cell types, the open signals obtained by batch ATAC-seq are a "mixture" of various cell types, potentially masking crucial but subtle epigenetic differences between different cell subpopulations. Single-cell ATAC-seq overcomes this bottleneck, allowing researchers to observe the "on / off" state of chromatin in each cell individually, thereby revealing cellular heterogeneity and identifying new cell subtypes or transitional cells that were previously averaged out.

[0003] The standard single-cell ATAC-seq procedure typically includes the following key steps: First, rapid isolation of cell nuclei from fresh tissue or cultured cells; second, Tn5 transposase reaction of the nuclei in an optimized buffer system to fragment open chromatin regions and ligate adapters; subsequently, PCR amplification to enrich effective library fragments; and finally, high-throughput sequencing and bioinformatics analysis of the library. High-quality ATAC-seq data typically exhibit significant enrichment signals near the transcription start site (TSS), a characteristic that can be quantified using the "TSSenrichment Score"—a higher score indicates a chromatin structure closer to physiological conditions and more reliable data quality.

[0004] Although single-cell ATAC-seq performs excellently in fresh tissue and cell samples, in practical clinical and research applications, a large number of biological samples are preserved long-term in frozen form (e.g., in liquid nitrogen or at -80°C). Therefore, developing a single-cell ATAC-seq detection method suitable for frozen tissues is of significant practical importance. However, existing studies have shown that directly extracting cell nuclei from frozen tissues for traditional ATAC-seq often leads to chromatin structure damage, thus affecting the quality of sequencing data.

[0005] Specifically, multiple independent research teams observed a common problem when using frozen tissue for single-cell ATAC-seq: the transcription start site enrichment score (TSSenrichment score) was significantly lower than that of fresh tissue samples. For example, in different types of samples, such as brain tissue, liver, and tumor tissue, the TSS enrichment score of ATAC-seq data after freezing generally decreased by more than 50%, severely affecting the accurate interpretation of chromatin open states. In addition, other quality control indicators, such as the fraction of reads in peaks (FRiP), often showed abnormalities, indicating impaired overall chromatin structural integrity.

[0006] In summary, existing single-cell ATAC-seq technology faces significant technical bottlenecks when processing frozen tissues, particularly in the decrease of TSS enrichment score, which limits its widespread application in clinical archives, biobanks, and other scenarios. Therefore, a novel detection method that can effectively preserve the original physiological state of chromatin in frozen tissues is urgently needed. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a sample processing method for detecting single-cell chromatin accessibility and its application, developing a chromatin detection method particularly suitable for cryopreserved tissue samples, aiming to improve the quality of ATAC-seq data and enhance the accuracy and reliability of chromatin accessibility detection.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a sample processing method for detecting single-cell chromatin accessibility, the sample processing method comprising:

[0010] The tissue sample is sliced ​​to obtain tissue sections, which are then subjected to permeabilization, transposase treatment, and cell nucleus extraction in sequence; the tissue sample includes frozen tissue samples.

[0011] This invention presents an improved method for detecting open chromatin regions, particularly suitable for cryopreserved tissue samples. It breaks through the traditional ATAC-seq paradigm of "nucleus extraction followed by fragmentation," innovatively integrating frozen tissue embedding, slicing, and in situ transposase reaction into a continuous process. This significantly improves the quality of ATAC-seq data, especially in key quality assessment indicators—the enrichment fraction of transcription start sites and the proportion of fragments overlapping with peaks.

[0012] Optionally, the step of OCT embedding is further included before the slicing process.

[0013] Optionally, the OCT embedding process includes mixing the tissue sample with an OCT embedding agent.

[0014] Optionally, the thickness of the tissue section is 3 to 20 μm, for example, it can be 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18 or 19 μm, preferably 5 to 10 μm.

[0015] Optionally, the permeabilization process includes: incubating the tissue slices with a permeabilization buffer.

[0016] Optionally, the permeation buffer contains a surfactant.

[0017] Optionally, the surfactant includes Tween and / or IGEPEAL CA-630.

[0018] Optionally, the volume percentage of the surfactant in the permeation buffer is 0.1% to 0.3%.

[0019] Optionally, the temperature for the mixed incubation is 2~6℃, for example, 3, 4 or 5℃, and the time is 10~20 minutes.

[0020] Optionally, the transposase treatment includes: mixing and incubating the permeabilized tissue slices with a transposase solution.

[0021] Optionally, the transposase solution contains a Tn5 transposase with a preloaded adapter.

[0022] Optionally, the temperature for the mixed incubation is 37~50℃, for example, 38, 39, 40, 45, 46, 47, 48 or 49℃, and the time is 30~60 min, for example, 35, 40, 45, 50 or 55 min.

[0023] Secondly, the present invention provides a high-throughput single-cell accessibility chromatin sequencing method, the sequencing method comprising:

[0024] The sample was processed using the sample processing method described in the first aspect to obtain cell nuclei. The cell nuclei were dispersed in a parallel microreaction system to add different cell tags to the chromatin DNA. All cell nuclei were then mixed and PCR amplification was performed using successfully transposable open chromatin as a template to construct a library. The library was then sequenced and analyzed using bioinformatics.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] This invention designs an "in situ transposition reaction" strategy, which effectively maintains the original spatial conformation and physiological state of chromatin in vivo, avoids chromatin structural disturbance caused by premature release of the cell nucleus during thawing, significantly improves the quality of ATAC-seq data, significantly improves the enrichment fraction of transcription start sites and the proportion of fragments overlapping with peaks, and provides clearer nucleosome localization signals, thereby improving the accuracy and reliability of chromatin openness detection. Attached Figure Description

[0027] Figure 1 This diagram shows a comparison between the novel chromatin accessibility detection process of the present invention and the traditional process.

[0028] Figure 2 This is a comparison diagram of chromatin structural integrity under different treatment conditions according to the present invention.

[0029] Figure 3 A distribution map of genomic transposon fragment lengths for nuclear extraction detection procedures in fresh tissue.

[0030] Figure 4 A distribution map of genomic transposon fragment lengths for nuclear extraction and detection of quick-frozen tissues.

[0031] Figure 5 A distribution map of genomic transposon fragment lengths for the OCT cryo-embedding nuclear extraction detection process.

[0032] Figure 6 This is a graph showing the length distribution of genomic transposon fragments in the in situ transposon detection procedure for frozen sections.

[0033] Figure 7 This is a distribution map of genomic transposon fragment lengths in the OCT slice in situ transposon detection process.

[0034] Figure 8 Comparison of chromatin structural integrity under different permeation and transposition conditions.

[0035] Figure 9 Comparison of chromatin structural integrity under different slice thickness conditions.

[0036] Figure 10 The figure shows the effect of different concentrations of formaldehyde fixation on the enrichment fraction of TSS. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0039] This invention proposes an improved method for detecting open regions of chromatin, particularly suitable for cryopreserved tissue samples. This method breaks through the traditional ATAC-seq paradigm of "nucleus extraction followed by fragmentation," innovatively integrating frozen tissue embedding, slicing, and in-situ transposase reaction into a continuous process, thereby maximally maintaining the original spatial conformation and physiological state of chromatin in vivo. By directly treating tissue sections with Tn5 transposase, chromatin structural disturbances caused by premature release of the nucleus during thawing are avoided, significantly improving the quality of ATAC-seq data, especially demonstrating superior performance in key quality assessment indicators—the enrichment fraction of transcription start sites and the proportion of fragments overlapping with peaks.

[0040] The traditional view holds that the poor quality of ATAC-seq data from cryopreserved tissue samples is primarily due to the formation of tiny ice crystals during freezing. These ice crystals melt upon thawing, causing drastic local water migration and leading to an imbalance in osmotic pressure inside and outside the cell. This "freeze-thaw stress" can cause cell membrane rupture, organelle swelling, and even localized damage to the nuclear membrane, thereby disrupting the spatial arrangement and higher-order structure of chromatin. However, this invention unexpectedly discovered in experiments that performing drastic procedures such as sonication, grinding, and nucleosome extraction on cell and tissue samples after transposase disruption does not affect the chromatin integrity detection results. Therefore, through rigorous design, we conducted transposase fragmentation experiments on samples with different treatments (see Example 1). We found that the step with the greatest impact on chromatin integrity was nucleus extraction after thawing. This was reflected in the fact that the TSS enrichment score of samples extracted after conventional thawing was only 38% of that of samples extracted from fresh tissue (4.09 vs 10.85), while the TSS enrichment score of frozen tissue after in-situ transposition on sections and then extraction reached 8.42, which was 78% of that of fresh tissue (8.42 vs 10.85). We further found that using OCT (optimal cutting temperature compound) to embed frozen tissue combined with in-situ transposition on sections could improve the TSS enrichment score to a certain extent, reaching 10.60, which is very close to the chromatin integrity index of fresh tissue. We further tested the percentage of fragments overlapping with peaks (FRiP) under different treatment conditions, which showed a similar trend to the TSS enrichment score. Figure 2 )

[0041] Furthermore, key optimization conditions for tissue section permeation and in situ transposition were disclosed, such as the type and concentration of surfactant, and the temperature and time for in situ transposition. Figure 8 ).

[0042] Furthermore, compared to a thickness of 50 μm, the tissue sections used in the detection process of this invention provide the best chromatin integrity and a good number of high-quality DNA fragments at a thickness of 5–20 μm. Figure 9 Furthermore, it can be applied to tissue sections fixed with 0.1% to 1% formaldehyde. Since formaldehyde fixation is a commonly used technique in tissue biology, this greatly broadens the application scenarios of this invention. Figure 10 )

[0043] This invention is not only applicable to conventional cryoembedded tissues, but can also be extended to clinical pathology archives, biobank samples, and other applications that require high chromatin integrity, thus having broad scientific research and clinical translational value.

[0044] In specific embodiments of the present invention, the following operating methods may be adopted.

[0045] 1. Sample preparation

[0046] Fresh tissues were rapidly embedded in OCT compounds or directly wrapped in aluminum foil and flash-frozen in liquid nitrogen for long-term storage at -80°C.

[0047] The tissue was cut into 5-20 μm thick sections using a cryostat at -20°C and attached to a detachable glass slide.

[0048] 2. Section fixation and permeabilization

[0049] Fix the sections with 0.1%~1% paraformaldehyde (PFA) at room temperature for 10 min (optional, to enhance structural stability).

[0050] After washing with PBS, add permeation buffer (containing 0.1%~0.3% IGEPAL CA-630) and incubate at 4°C for 10 min.

[0051] 3. In situ transposase reaction

[0052] Remove the permeabilization solution and add the ATAC-seq transposase mixture (containing Tn5 transposase preloaded adapter, buffer, Mg). 2+ DMF).

[0053] React at 37~50℃ for 30~60 min.

[0054] The reaction was terminated by replacing the solution with a stop solution (containing 10 mM EDTA).

[0055] 4. Extract cell nuclei

[0056] Tissues are physically disrupted using a tissue homogenizer (such as a Dounce glass homogenizer, electric homogenizer, or ultrasonic homogenizer) in a nuclear separation buffer containing a nonionic detergent (such as 0.1%~0.5% NP-40, Tween20, etc.) to selectively dissolve cell membranes and release cell nuclei.

[0057] Cell nuclei were obtained by removing cell debris through low-speed centrifugation.

[0058] 5. Library construction and sequencing

[0059] Sequencing libraries can be constructed using commercially available ATAC-seq or high-throughput single-cell ATAC-seq kits, such as SeekOne® DD high-throughput single-cell ATAC+RNA kit or 10×genomics ATAC-seq kit.

[0060] Perform sequencing according to the requirements of the sequencing platform, such as the second-generation sequencing platforms of companies like Illumina and Genemax.

[0061] Example 1

[0062] This invention validates the ATAC-seq method based on in-situ transposition of frozen tissue sections.

[0063] Sample source and reagents

[0064] Samples: Brain tissue from C57BL / 6 mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.). After fresh collection, a portion of the tissue was directly extracted for nucleus extraction and labeled as "fresh tissue nucleus extraction". The remaining brain tissue was immediately flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0065] Reagents: SeekOne ATAC+RNA dual-omics reagent (Beijing Xunyin Biotechnology Co., Ltd.), used for ATAC library construction; OCT embedding agent (Sakura Tissue-Tek OCT Compound) used for tissue cryoprotection.

[0066] Experimental steps

[0067] 1. Sample preparation and cryosectioning

[0068] Tissue processing: Mouse brain tissue blocks flash-frozen in liquid nitrogen were immersed in OCT embedding medium, pre-cooled at -80℃ and then flash-frozen to form a solid shape, and labeled as "OCT embedded"; unembedded liquid nitrogen flash-frozen samples were labeled as "frozen tissue".

[0069] "Fresh tissue nucleus extraction" samples were used to construct a high-throughput single-cell ATAC-seq library using SeekOne ATAC+RNA dual-omics reagent.

[0070] Traditional ATAC library construction: Cell nuclei are directly extracted from a portion of "frozen tissue" and "OCT-embedded tissue" and high-throughput single-cell ATAC-seq libraries are constructed using SeekOne ATAC+RNA dual-omics reagents.

[0071] The other part of the "frozen tissue" and "OCT embedded tissue" were sectioned as follows.

[0072] Sectioning procedure: The tissue was sectioned to a thickness of 10 μm using a Leica CM1950 cryostat (-20℃ environment) and attached to a clean glass slide (Thermo Fisher Superfrost Plus).

[0073] 2. In situ transposase reaction and library construction

[0074] Permeabilization: After the sample was sliced, it was washed with PBS and then permeabilization buffer (containing 0.2% IGEPAL CA-630) was added and incubated at 4°C for 15 min.

[0075] Transposase reaction: Remove permeabilization solution, add transposase mixture from SeekOne ATAC kit (containing pre-loaded Tn5 transposase with adapter and Mg²⁺ buffer), and react at 37°C in the dark for 45 min.

[0076] Nucleus extraction from tissue sections: The reaction was terminated by adding a stop solution (containing 10 mM EDTA), and cell nuclei were extracted from the sections using an ultrasound device and counted for quality control.

[0077] Library construction: Perform water-in-oil microdroplet reaction according to the kit instructions, followed by PCR amplification (12-14 cycles) to finally construct a single-cell ATAC-seq library.

[0078] 3. Data Analysis and Result Validation

[0079] 3.1 Transcription start site enrichment score (TSS) analysis

[0080] Data alignment: Sequencing reads were aligned to the mm10 reference genome, and low-quality reads were filtered before Peakcalling (MACS2).

[0081] TSS enrichment calculation: Calculate the TSS enrichment score (normalized to the signal strength of the TSS ± ​​500 bp region).

[0082] result:

[0083] Traditional methods yielded TSS enrichment scores of 4.09 (direct nuclear extraction from quick-frozen tissue) and 5.66 (nucleus extraction from OCT cryo-embedding).

[0084] The TSS enrichment score of the method of this invention is 8.42 (in situ transposition in frozen sections) and 10.60 (in situ transposition in OCT sections), which is significantly higher than that of the traditional method and close to the fresh tissue index of 10.85, thus maintaining good chromatin structural integrity. Figure 2 ).

[0085] 3.2 Analysis of the percentage of reads in peaks (FRiP)

[0086] FRiP calculation: Statistical comparison of the percentage of Reads aligned to the Peak region.

[0087] result:

[0088] Traditional methods FRiP: 10.05% (direct nucleus extraction from quick-frozen tissue) and 15.38% (nucleus extraction from OCT cryo-embedding).

[0089] The method of this invention achieves FRiP rates of 20.57% (in situ transposition of frozen sections) and 21.89% (in situ transposition of OCT sections), with an average improvement of 67%. Figure 2 ).

[0090] 3.3 Verification of chromatin structure

[0091] Nucleosome localization signal: By statistically analyzing the lengths of genomic fragments disrupted by transposition, the method of this invention presents a clear periodic nucleosome signal in the TSS region (+1 nucleosome peak spacing approximately 147 bp), consistent with the length distribution of genomic transposition fragments in fresh tissue, while the signal is blurred using traditional methods. Figures 3-7 ).

[0092] This embodiment verifies the following key advantages by comparing the traditional frozen tissue single-cell ATAC-seq method with the "in situ transposition of frozen sections" strategy proposed in this invention:

[0093] TSS enrichment score improvement: In situ processing of frozen sections improved the TSS enrichment score from 4.09 in the traditional method to 10.60, which is close to the level of fresh tissue (fresh tissue TSS enrichment score ≈10.85).

[0094] FRiP is significantly improved: the effective signal ratio increases from 10.05% to 21.38%, and background noise is significantly reduced.

[0095] Chromatin structural integrity: The periodic signals of nucleosomes are clear and the technical reproducibility is excellent, proving that the native state of chromatin is preserved.

[0096] Example 2

[0097] This embodiment optimizes tissue permeation and transposition conditions based on the in situ transposition method of frozen tissue sections.

[0098] This embodiment focuses on OCT-embedded mouse brain frozen sections. By adjusting the tissue permeation reagent and concentration, transposition temperature and time, the optimal conditions were selected by comprehensively considering three indicators: the yield of cell nuclei after in situ transposition, the number of high-quality DNA fragments in the median cell nucleus, and the enrichment efficiency of transcription start site (TSS).

[0099] Experimental steps

[0100] 1. Sample preparation

[0101] Mouse brain tissue was flash-frozen in liquid nitrogen, embedded using OCT, and prepared into continuous frozen sections with a thickness of 10 μm. These sections were then attached to poly-L-lysine-coated glass slides and stored at -80°C for later use (to maintain tissue morphology and molecular integrity).

[0102] 2. Permeation treatment and transposition treatment

[0103] Permeabilization: Prepare PBS buffers containing different concentrations of surfactant according to the conditions in Table 1, place the frozen sections in the above different PBS buffers, and incubate at 4°C for 15 min.

[0104] Table 1

[0105]

[0106] Transposition: Remove the permeabilization solution and add the transposase mixture from the SeekOne ATAC kit (containing pre-loaded Tn5 transposase with adapters, Mg...). 2+ (Buffer solution), react at 37°C or 50°C in the dark for 60 min.

[0107] Nucleus extraction from tissue sections: The reaction was terminated by adding a stop solution (containing 10 mM EDTA), and cell nuclei were extracted from the sections using an ultrasound device and counted for quality control.

[0108] Library construction: Perform water-in-oil microdroplet reaction according to the kit instructions, followed by PCR amplification (12-14 cycles) to finally construct a single-cell ATAC-seq library.

[0109] Results Analysis

[0110] like Figure 8 As shown, conditions 1-7 showed varying performance in terms of nucleus yield, median number of high-quality DNA fragments in the nucleus, and transcription start site (TSS) enrichment efficiency. Conditions 2, 4, and 6 showed the best overall performance, especially condition 6, where the transposase reaction temperature of 50℃ significantly increased the median number of high-quality DNA fragments (4925 vs 3641) compared to 37℃ (condition 4). Condition 5, using 0.1% Triton X-100, significantly reduced the number of extracted nuclei (19650 vs 85050) and the transcription start site enrichment fraction (6.25 vs 9.58) compared to condition 4. While condition 7, without permeabilization, yielded the highest number of nuclei, it only produced a very small number of high-quality DNA fragments in the median (433), indicating that permeabilization is essential.

[0111] Example 3

[0112] This embodiment tests the effect of tissue section thickness on the ATAC-seq detection effect of in situ transposition.

[0113] Using mouse brain tissue as a sample, single-cell ATAC-seq of OCT slices of different thicknesses (5 μm, 10 μm, 20 μm, 50 μm) was performed according to the procedure in condition 4 of Example 2. The results are as follows. Figure 9 As shown.

[0114] Results analysis:

[0115] The number of high-quality fragments: 5 μm (3599) and 10 μm (3516) slices were significantly higher than 20 μm (2871) and 50 μm (2046), indicating that thin slices are more conducive to Tn5 enzyme penetration and capture sufficient chromatin opening signals;

[0116] TSS enrichment scores of 5 μm (9.14) and 10 μm (9.28) were significantly higher than those of 20 μm (8.23) and 50 μm (7.19), indicating that open chromatin signals in thin sections are more strongly associated with gene transcription regulation and have higher detection specificity.

[0117] This embodiment demonstrates, by comparing the in situ transposition detection results of OCT slices of different thicknesses, that 5~10 μm is the optimal thickness range for detecting chromatin accessibility in OCT embedded slices via in situ transposition, which can simultaneously take into account signal intensity (number of high-quality fragments) and specificity (TSS enrichment).

[0118] Example 4

[0119] This embodiment tests the effect of formaldehyde fixation concentration on the in situ transposition TSS enrichment of OCT-embedded tissue sections.

[0120] This embodiment compares the in situ transposition effect of OCT-embedded tissue sections under different formaldehyde concentrations to determine whether the in situ transposition detection process of the present invention is suitable for formaldehyde-fixed tissue sections.

[0121] Using mouse brain tissue as a sample, single-cell ATAC-seq of OCT tissue sections fixed with different formaldehyde concentrations was performed according to the procedure in condition 4 of Example 2. The results are as follows. Figure 10 As shown.

[0122] Results analysis:

[0123] TSS enrichment scores: The TSS enrichment scores of tissue sections fixed with 0.1% formaldehyde (8.73) and 1% formaldehyde (8.67) were slightly lower than those of unfixed tissue sections (9.38), while tissue sections fixed with 4% formaldehyde showed a significant decrease in TSS enrichment scores, indicating that the in situ transposition detection procedure of the present invention can be adapted to tissue sections fixed with low concentrations of 0.1-1% formaldehyde.

[0124] In summary, this invention provides a novel strategy for transposase-accessible chromatin sequencing of frozen tissues. It avoids extracting the cell nucleus before the transposase breaks down the genomic DNA. Instead, it directly performs low-temperature slicing of frozen tissues (whether or not OCT-embedded) and in-situ permeabilization and reaction with Tn5 transposase on the tissue slices. This solves the long-standing problem of protecting the chromatin structure of frozen samples in the field of epigenomics, significantly improving the quality of ATAC-seq data. Furthermore, by optimizing the reaction conditions, the data quality is further improved, achieving a level close to or even reaching that of detection using fresh tissues.

[0125] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A sample processing method for detecting single-cell chromatin accessibility, characterized in that, The sample processing method includes: The tissue sample was sliced ​​to obtain tissue sections, and the tissue sections were sequentially permeabilized, treated with transposase, and had cell nuclei extracted. The tissue samples include frozen tissue samples.

2. The sample processing method for detecting single-cell chromatin accessibility according to claim 1, characterized in that, The step of OCT embedding is also included before the slicing process; Optionally, the OCT embedding process includes mixing the tissue sample with an OCT embedding agent.

3. The sample processing method for detecting single-cell chromatin accessibility according to claim 1 or 2, characterized in that, The thickness of the tissue sections is 3~20 μm, preferably 5~10 μm.

4. The sample processing method for detecting single-cell chromatin accessibility according to any one of claims 1-3, characterized in that, The permeation treatment includes: mixing the tissue slices with permeation buffer and incubating them.

5. The sample processing method for detecting single-cell chromatin accessibility according to claim 4, characterized in that, The permeation buffer contains a surfactant; Optionally, the surfactant includes Tween and / or IGEPEAL CA-630; Optionally, the volume percentage of the surfactant in the permeation buffer is 0.1% to 0.3%.

6. The sample processing method for detecting single-cell chromatin accessibility according to claim 4, characterized in that, The temperature for the mixed incubation is 2~6℃, and the time is 10~20 min.

7. The sample processing method for detecting single-cell chromatin accessibility according to claims 1-5, characterized in that, The transposase treatment includes: mixing and incubating the permeabilized tissue slices with a transposase solution.

8. The sample processing method for detecting single-cell chromatin accessibility according to claim 7, characterized in that, The transposase solution contains Tn5 transposase with a preloaded adapter.

9. The sample processing method for detecting single-cell chromatin accessibility according to claim 7, characterized in that, The temperature for the mixed incubation is 37~50℃, and the time is 30~60 min.

10. A high-throughput single-cell accessibility chromatin sequencing method, characterized in that, The sequencing method includes: The sample is processed using the sample processing method according to any one of claims 1-9 to obtain cell nuclei. The cell nuclei are dispersed in a parallel microreaction system to add different cell tags to the chromatin DNA. All cell nuclei are then mixed and PCR amplification is performed using successfully transposable open chromatin as a template to construct a library. The library is then sequenced and subjected to bioinformatics analysis.

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