Method for improving accessibility of chromatin DNA in cells and application of method

Abstract

The invention discloses a method for improving accessibility of chromatin DNA in cells and application of the method, and relates to the technical field of gene sequencing and tissue cell sample analys.The method comprises the following steps that a sample to be detected is provided; the method comprises the following steps: mixing a to-be-detected sample with an alcohol reagent to permeate cells and expose a heterochromatin region of the to-be-detected sample to obtain a pretreated sample; carrying out fragmentation treatment on the DNA of the pretreated sample, carrying out reverse transcription reaction on the RNA in the pretreated sample, marking the fragmented DNA and the product cDNA after the reverse transcription reaction, and separating the marked genome DNA and cDNA; and library construction and sequencing are respectively carried out on the marked genome DNA fragments and cDNA products to obtain single cell genomics information and transcriptome information in the sample to be detected, the alcohol reagent can open heterochromatin without deviation to obtain more nucleic acid fragments, the single cell genome detection sensitivity is improved, RNA detection is not influenced, and the method is simple and convenient to operate. The biomics analysis of the high-flux single cell whole genome DNA and RNA is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of gene sequencing and tissue cell sample analysis, and in particular to a method for improving the accessibility of chromatin DNA in cells and an application thereof. Background Art

[0002] High-throughput single-cell sequencing technologies enable the parallel analysis of molecular features across thousands of cells, making them invaluable for analyzing cellular heterogeneity. These technologies cover multiple dimensions, including the transcriptome, chromatin accessibility, copy number variation (CNV), somatic mutations, and DNA methylation. Single-cell RNA sequencing (scRNA-seq) has become a routine analytical tool due to its technical maturity. However, large-scale application of single-cell genomic analysis has yet to be achieved. This technical bottleneck stems primarily from two key factors: first, each cell contains only two copies of genomic DNA; second, the majority of genomic DNA exists as highly compacted heterochromatin. These characteristics make efficient labeling of single-cell DNA challenging, resulting in generally low genomic coverage. For example, when single-cell chromatin accessibility sequencing (scATAC-seq) is performed on human peripheral blood mononuclear cells (PBMCs) directly treated with Tn5 transposase, only 10,000–20,000 DNA fragments are obtained per cell, representing only approximately 0.1% of the entire genome.

[0003] To improve genomic coverage and ensure consistent cell labeling of homologous DNA, researchers have developed a variety of innovative approaches. Mission Bio's Tapestri platform, based on microfluidics, employs a two-step water-in-oil-in-oil system for cell capture and labeling. The first step, water-in-oil encapsulation, encapsulates cells and proteases in microdroplets, fully releasing genomic DNA; the second step, water-in-oil, completes cell-specific DNA barcoding. Similarly, 10X Genomics' single-cell DNA copy number detection technology also employs a two-step water-in-oil strategy, but the first encapsulation step immobilizes DNA in hydrogel beads rather than microdroplets. While these approaches enable single-cell DNA labeling, they are limited by high reagent costs and time-consuming procedures. Using individual cells as natural reaction vessels and enhancing transposase cleavage efficiency through other methods has successfully achieved high-throughput single-cell DNA methylation detection. However, this approach is hampered by complex cell pretreatment procedures, the risk of cross-contamination of intercellular DNA, and limited genomic coverage due to insufficient sensitivity. These technical challenges highlight the need for further methodological breakthroughs in single-cell genomic analysis. Summary of the Invention

[0004] The main purpose of the present invention is to propose a method for improving the accessibility of chromatin DNA in cells and its application, aiming to solve the risk of cross-contamination of DNA between cells and the problem of limited genome coverage due to insufficient sensitivity during the processing using existing high-throughput single-cell technology.

[0005] To achieve the above object, the present invention provides a method for improving the accessibility of chromatin DNA in cells, comprising the following steps: S10, providing a sample to be tested; S20, mixing the sample to be tested with an alcohol reagent to permeabilize cells and expose the heterochromatin region of the sample to be tested, to obtain a pretreated sample; S30, fragmenting the DNA of the pretreated sample to obtain fragmented DNA, performing reverse transcription on the RNA in the pretreated sample to obtain cDNA products, labeling the fragmented genomic DNA and the cDNA products, and separating the labeled genomic DNA and the labeled cDNA products; S40, constructing a genomic library and a transcriptome library for the labeled genomic DNA fragments and the labeled cDNA products, respectively, and sequencing them to obtain single-cell genomic information and transcriptome information in the sample to be tested.

[0006] In one embodiment, in step S20, the alcohol reagent includes at least one of methanol, ethanol and isopropanol.

[0007] In one embodiment, the volume concentration of the alcohol reagent is 50-100%; and / or, The permeabilization temperature is -80°C to 25°C; and / or, In one embodiment, the permeabilization treatment time is 0.08h~24h.

[0008] In step S30 , the reagents used in the fragmentation process include transposase or endonuclease.

[0009] In one embodiment, in step S30, the step of labeling the genomic DNA includes: Provide labels; The tags are added to the fragmented DNA by polymerase extension and / or ligase ligation.

[0010] In one embodiment, the carrier of the tag is a polymer microbead or a nano DNA microsphere; and / or, The labels on the same carrier are the same, and the labels on different carriers are different.

[0011] In one embodiment, in step S30, the step of labeling the cDNA product comprises: S301, performing a reverse transcription reaction on the RNA in the pretreated sample to obtain a cDNA product; S302 , providing a tag sequence, and connecting the tag sequence to the cDNA product through a reverse transcription reaction to label the cDNA product.

[0012] In one embodiment, in step S40, the separation method includes nucleic acid affinity purification, antibody or streptavidin enrichment, or PCR amplification enrichment.

[0013] The present invention also provides a sequencing library, which includes a sequencing library constructed by any one of the above methods for improving the accessibility of chromatin DNA in cells.

[0014] The present invention also provides an application of a sequencing library constructed by the method for improving the accessibility of chromatin DNA in cells as described in any of the above items in high-throughput single-cell genome epigenetic modification detection.

[0015] The present invention destroys the higher-order structure of the cell nuclear membrane and chromatin by mixing alcohol reagents with the sample to be tested, fully exposing the heterochromatin region, thereby avoiding the damage to the sample caused by complex steps such as traditional fixation, termination and permeabilization. It not only simplifies the experimental process and reduces operation time and human error, but also maximizes the preservation of cell morphological integrity and intracellular nucleic acids on the basis of ensuring cell integrity; alcohol reagents have multiple functions of removing nucleosomes, permeabilization and dehydration, providing a more uniform substrate background for subsequent fragmentation processing. Since the heterochromatin region is opened without bias, the cutting efficiency is significantly improved, more nucleic acid fragments are obtained and more complete nucleic acid information is retained, thereby improving the sensitivity and coverage of high-throughput single-cell genome detection. In addition, alcohol treatment does not affect RNA detection, enabling high-throughput dual-omics co-detection of whole-genome DNA and RNA at the single-cell level, thereby realizing high-throughput co-detection of DNA and RNA in the same cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0017] Figure 1Schematic diagram of quality control of DNA fragment distribution after Tn5 fragmentation of K562 cell nuclei treated with or without methanol according to an embodiment of the present invention; Figure 2 Schematic diagram showing comparison of the quality control indicators transcription start site (TSS) and fraction fragments overlapping peaks (Fraction Fragments overlapping Peaks) of high-throughput single-cell nucleus DNA sequencing of K562 cells treated with or without methanol in one embodiment of the present invention; Figure 3 Schematic diagram comparing quality control indicators of high-throughput single-cell nucleus DNA sequencing of K562 cells treated with or without methanol in one embodiment of the present invention.

[0018] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0020] To improve genome coverage and ensure consistent cell labeling for homologous DNA, researchers have developed a variety of innovative solutions. Mission Bio's Tapestri platform, based on microfluidics, uses a two-step water-in-oil-in-oil system to capture and label cells: the first step, water-in-oil encapsulation, encapsulates cells and proteases in microdroplets, allowing the genomic DNA to be fully released; the second step, water-in-oil, completes the cell-specific barcoding of the DNA. Similarly, 10X Genomics' single-cell DNA copy number detection technology also uses a two-step water-in-oil strategy, but its first encapsulation step is to fix the DNA in hydrogel microbeads rather than microdroplets. Although this type of method can achieve DNA labeling at the single-cell level, it still has limitations such as high reagent costs and time-consuming operations.

[0021] The sci-MET technique developed by Andrew C. Adey's team takes a different approach, using single cells as natural reaction vessels. By loosening chromatin through treatment with sodium dodecyl sulfate (xSDS) or lithium diiodosalicylate (LAND), the technique enhances transposase cleavage efficiency and successfully enables high-throughput single-cell DNA methylation detection. However, this method is limited by complex cell pretreatment procedures, the risk of cross-contamination of DNA between cells, and insufficient sensitivity leading to limited genomic coverage. These technical challenges highlight the need for further methodological breakthroughs in single-cell genomic analysis.

[0022] In view of this, the present invention provides a method for improving the accessibility of chromatin DNA in cells and its application, comprising the following steps: S10, providing a sample to be tested; S20, mixing the sample to be tested with an alcohol reagent to permeabilize cells and expose the heterochromatin region of the sample to be tested, to obtain a pretreated sample; S30, fragmenting the DNA of the pretreated sample to obtain fragmented DNA, performing reverse transcription on the RNA in the pretreated sample to obtain cDNA products, labeling the fragmented genomic DNA and the cDNA products, and separating the labeled genomic DNA and the labeled cDNA products; S40, constructing a genomic library and a transcriptome library for the labeled genomic DNA fragments and the labeled cDNA products, respectively, and sequencing them to obtain single-cell genomic information and transcriptome information in the sample to be tested.

[0023] In the present invention, by mixing an alcohol reagent with the sample to be tested, the nuclear membrane and higher-order chromatin structure of the sample to be tested can be destroyed, and the heterochromatin region of the sample to be tested can be fully exposed, thereby avoiding damage to the sample to be tested during the complex fixation, termination and permeabilization steps used in the prior art. This not only simplifies the experimental process, but also reduces operation time and potential human errors. The alcohol reagent used has the functions of removing nucleosomes, permeabilization and dehydration, and can also retain nucleic acids in cells to the maximum extent while ensuring cell integrity, while maintaining cell morphological integrity. In addition, the pre-treated sample is then fragmented. Since alcohol treatment can open heterochromatin without deviation, the cutting efficiency during the fragmentation process is significantly improved, so that more nucleic acid fragments can be obtained, nucleic acid information is retained, thereby improving the sensitivity and coverage of high-throughput single-cell genome detection. Furthermore, the treatment with the alcohol reagent does not affect RNA detection, which can break through the traditional method's inability to achieve co-detection of DNA and RNA in the whole genome of a single cell, thereby achieving high-throughput co-detection of DNA and RNA in the same cell.

[0024] It should be noted that compared with traditional ATAC-seq (which relies on physiologically open chromatin), the present invention can achieve random cutting of the entire genome through physical destruction, avoiding dependence on the transcription start site (TSS). It is suitable for cell types enriched in heterochromatin (such as neurons and tumor stem cells), breaking through the limitation of traditional methods that cannot achieve co-detection of DNA and RNA in the entire genome of a single cell.

[0025] In some embodiments, in step S20, the alcohol reagent includes at least one of methanol, ethanol and isopropanol. The alcohol reagent used has the functions of removing nucleosomes, permeabilization and dehydration, and can retain nucleic acids in cells to the maximum extent while ensuring cell integrity, while maintaining cell morphological integrity.

[0026] In some embodiments, the volume concentration of the alcohol reagent is 50-100%, that is, the volume concentration of the alcohol reagent can be 50%, 60%, 70%, 80%, 90% or 100%, and more preferably 70-100%. For example, 75% methanol means adding 75 mL of methanol to a 100 mL measuring cylinder. Within the above concentration range, the alcohol reagent can fully expose and open the chromatin structure in the cell nucleus of the sample to be processed, so that more nucleic acid fragments can be provided subsequently, thereby improving the sensitivity of sequencing.

[0027] In some embodiments, in step S20: the mixing temperature is -80°C~25°C; it can be any temperature in the above temperature range. Within this temperature range, the sample can be processed in a low temperature environment, which can effectively reduce enzyme activity, thereby preventing the degradation of DNA, RNA and protein, and also help maintain the original methylation pattern, avoiding false positive or false negative results caused by temperature changes, and can better protect the integrity of the sample and simplify the experimental process.

[0028] In some embodiments, the mixing time is 0.08 h to 24 h. Within the above time range, the sample to be tested and the alcohol reagent can be mixed to fully react.

[0029] In some embodiments, in step S30, the reagents used for nucleic acid fragmentation include transposase or endonuclease treatment. During the aforementioned alcohol-based treatment, while maintaining the integrity of the nucleus, the Tn5 transposase can accurately fragment DNA in non-open regions of the genome within the nucleus that would otherwise be inaccessible, thereby significantly increasing the detection rate of single-cell genomic fragments. Specifically, the transposase includes Tn5 transposase, and the endonuclease includes MboI.

[0030] Furthermore, in step S30, the step of labeling the fragmented genomic DNA includes: Provide labels; The tags are added to the fragmented DNA by polymerase extension and / or ligase ligation. The tags refer to short sequences (DNA or RNA fragments) that are used to uniquely identify each sample or cell. They are designed to ensure that the origin of each molecule or cell can still be traced even after all samples are mixed and processed together.

[0031] In some embodiments, the carrier of the label is a polymer microbead or a nano-DNA microsphere. The use of a label carrier helps ensure that each molecule or cell can be accurately labeled, thereby reducing data confusion caused by cross-contamination between samples. For example, nano-DNA microspheres have extremely high sequence specificity, which means that each nano-DNA microsphere can be encoded by a unique DNA sequence, ensuring accurate identification and differentiation even in complex samples.

[0032] In some embodiments, the labels on the same carrier are the same, and the labels on different carriers are different.

[0033] It should be noted that nucleic acid molecules derived from different cell sources are marked with different labels, and nucleic acid molecules derived from the same cell source are marked with the same label or labels with a specific correlation; DNA nucleic acid molecules and RNA nucleic acid molecules derived from the same cell can be marked with the same label or labels with a specific correlation, so that they can be distinguished.

[0034] In one embodiment, in step S30, the step of labeling the cDNA product comprises: S301, performing a reverse transcription reaction on the RNA in the pretreated sample to obtain a cDNA product; S302 , providing a tag sequence, and connecting the tag sequence to the cDNA product through a reverse transcription reaction to label the cDNA product.

[0035] It is understandable that in order to prevent RNA degradation and ensure that RNA information can be accurately captured during subsequent processing, alcohol reagents can be used as protective agents. Alcohols can fix intracellular components without damaging the integrity of RNA, thereby providing stability for subsequent separation steps. In addition, the label sequence can be selected as a reverse transcription primer with a label sequence of a cell barcode, and the label sequence can be added during the reverse transcription reaction, or the label sequence can be added to the cDNA product through a ligation reaction without a cell barcode.

[0036] In some embodiments, in step S40, the separation method includes nucleic acid affinity purification, antibody or streptavidin enrichment, or PCR amplification enrichment.

[0037] Nucleic acid affinity purification is a technique for purifying nucleic acids based on the specific interaction between nucleic acids and specific ligands. For example, probes (such as oligonucleotides) can be designed to specifically bind to target nucleic acid sequences and then used to isolate the target nucleic acid from a complex mixture. This method allows for highly specific capture of nucleic acid fragments of interest.

[0038] Antibody enrichment: Commonly used for protein studies, it can also be applied to nucleic acids tagged with specific antigenic epitopes. By using antibodies against these epitopes, the target molecule can be selectively enriched from a complex mixture.

[0039] Streptavidin enrichment: Based on the extremely high affinity between biotin and streptavidin, if the target nucleic acid or its tag is labeled with biotin, it can be enriched using media such as streptavidin (or avidin)-coated magnetic beads. This method is very common in genomic research, particularly for immobilizing and purifying DNA fragments with specific adapters when constructing sequencing libraries.

[0040] PCR amplification and enrichment is a widely used molecular biology technique for amplifying specific DNA fragments. By designing specific primers, the target region of interest can be selectively amplified, effectively enriching it. This method is not limited to genomic DNA but also applies to cDNA (reverse transcribed from RNA), making it equally important in transcriptome analysis.

[0041] In some embodiments, in step S40, the step after separating the labeled multiple nucleic acid fragments further includes constructing an epigenetic modification library to obtain epigenetic change information of single-cell genomics in the sample to be tested. Specifically, using the library obtained by the present invention, due to the improved sensitivity and coverage, a single-cell DNA base modification detection library can be constructed through biochemical reactions, such as DNA-5mC methylation and 5hmC hydroxymethylation detection library, so that high-throughput single-cell genome epigenetic modification detection can be achieved subsequently.

[0042] The present invention also provides a sequencing library, comprising a sequencing library constructed by any one of the above-mentioned methods for improving the accessibility of chromatin DNA in a cell, wherein the sequencing library has all the technical solutions of the method for improving the accessibility of chromatin DNA in a cell and the method for improving the accessibility of chromatin DNA in a cell, and thus has all the beneficial effects of the method for improving the accessibility of chromatin DNA in a cell and the method for improving the accessibility of chromatin DNA in a cell, and the present invention will not be repeated here one by one.

[0043] It should be noted that sequencing can be performed using Illumina, BGI or Zhenmai sequencers and all sequencing platforms that are compatible with the sequencing library of the present invention. Ordinary technicians in this field can modify the sequencing primer sequences in the sequencing library according to the requirements of different sequencing platforms to adapt to different sequencing platforms. These changes do not affect the technical protection scope of this patent.

[0044] The present invention also provides an application of a sequencing library constructed by the method for improving the accessibility of chromatin DNA in cells as described above in high-throughput single-cell genome epigenetic modification detection, for example, a library construction kit can be prepared.

[0045] The raw materials of the following examples are derived from: K562 cell line: purchased from ATCC, ATCC number CRL-3344; Human PBMC cells: purchased from AllCells, catalog number PB003F-C; Commercial SeekOne® DD Single-Cell ATAC+RNA Dual-Omics Kit: purchased from Beijing SeekOne Biotechnology Co., Ltd., catalog number K02901.

[0046] Example 1: High-throughput single-cell genomic DNA and RNA co-detection application Step 1: Preparation of cell nucleus suspension and treatment with alcohol reagents 1.1 Take fresh K562 cells, add NIB nuclear extraction buffer and treat on ice for 5 minutes, centrifuge at 500g, resuspend in PBS buffer and adjust the cell density to 1×10 6 mL, such as Figure 1 As shown, it is marked as control sample C1; 1.2. Take 100 mL of sample C1 and add 500 mL of -20℃ pre-cooled methanol dropwise. Let it stand at -20℃ for 30 minutes. Centrifuge at 1000g to remove the methanol and resuspend it in PBS buffer. Figure 1 As shown, it is marked as experimental sample T1; Step 2: Prepare DNA and RNA libraries from nuclear samples C1 and T1, respectively, using the commercial SeekOne® DD Single Cell ATAC+RNA Dual-Omics Kit. The steps are summarized as follows: 2.1 Chromatin accessible region fragmentation: Genome fragmentation by Tn5 transposase; 2.2 Oil-in-water generation: Barcode beads are used to label the chromatin DNA accessible region sequence and RNA sequence in the same cell nucleus. RNA is labeled based on semi-random primer reverse transcription, and ATAC sequence is labeled based on ligation strategy. 2.3 Pre-amplification: Enrichment of labeled products (RNA sequences and DNA sequences); 2.4 Amplification and Library Construction: DNA libraries were directly amplified using Truseq Read1 SeqPrimer and Nextera Read2SeqPrimer primers. RNA libraries were amplified using Truseq Read1 SeqPrimer and template-switch primers followed by fragmentation and ligation. Step 3: Library quality control and sequencing 3.1 The library was screened with AMPure XP magnetic beads (150-800 bp fragments), analyzed using an Agilent TapeStation 4200 fragment analyzer, and quantified by Qubit before sequencing on an Illumina NovaSeq 6000 or SurfSeq 5000, both using the PE150 sequencing strategy. The test results are as follows: Figure 1 As shown in the figure, the C1 library that has not been treated with methanol presents a peak pattern with superimposed nucleosome fragment size (140 bp), which is a normal ATAC library; while the T1 cell nucleus library that has been treated with methanol has a uniform fragment distribution, indicating that the chromatin structure in the cell nucleus is opened.

[0047] 3.2 Data Analysis: SeekArc tools were used to perform UMI correction, alignment, and TSS quality control analysis. The analysis results are as follows: Figure 2 As shown, Figure 2 A in the figure shows that the TSS value of C1 in K562 cells that were not treated with methanol was 7.47. Figure 2 Figure B shows that the TSS value of the T1 nucleus treated with methanol is only 1.54. In addition, according to the index of the fraction ratio of overlapping peaks, Figure 2 The C in the figure is the index of C1 in K562 cells that have not been treated with methanol, which is 60.4%. Figure 2 D in the figure is only 1.9% for T1 treated with methanol. These two indicators prove that the use of methanol reagent destroys the chromatin structure, and the DNA that can be interrupted by Tn5 is no longer limited to the transposition start site (TSS) under physiological conditions, but is randomly interrupted on the genome, so there is no peak.

[0048] In addition, the number of DNA fragments detected in the median cells was further analyzed, and the test results were as follows: Figure 3As shown, the median cell of the methanol-treated T1 sample detected 75,017 fragments in 82,570 read pairs; while the normal cell nucleus C1 that was not treated with methanol only detected 12,367 fragments in 104,074 read pairs, a difference of 6 times. This proves that using alcohol treatment to increase the accessibility of heterochromatin DNA in the nucleus can significantly increase the average number of DNA fragments detected in single cells.

[0049] In addition, as shown in Table 1, analysis of the RNA library demonstrated that the method of treating the cell nucleus with methanol did not affect RNA detection.

[0050] Table 1: Comparison of core quality control indicators for high-throughput single-cell RNA sequencing of K562 cells with and without methanol treatment

[0051] Example 2: Application of high-throughput single-cell methylation detection using alcohol pretreatment Step 1: Preparation of PBMC nuclear suspension and differential treatment 1.1 Human PBMC cells were treated with 1× lysis buffer on ice for 3 min, centrifuged at 500 g, and resuspended in PBS buffer to adjust the cell density to 1×10 6 mL; 1.2 Take 100 mL of nuclear suspension and add dropwise 500 mL of -20°C pre-cooled 100% ethanol. Label this sample as experimental sample T2. Take another 100 mL of nuclear suspension and add dropwise 500 mL of -20°C pre-cooled 100% isopropanol. Label this sample as experimental sample T3. Both T2 and T3 are allowed to stand at -20°C for 30 minutes. Centrifuge at 1000 g to remove the ethanol or isopropanol and resuspend in PBS buffer. Take another 100 mL of nuclear suspension and treat the nuclei according to the xSDS protocol in the sci-MET technique, that is, first fix the nuclei with 1.5% formaldehyde and then use 0.3% SDS to remove nucleosomes from the nuclear chromatin. Label this sample as control sample C2. Step 2: Prepare single-cell methylation libraries from cell nucleus samples C2, T2, and T3 using the commercial SeekOne® DD Single-Cell Methylation Detection Kit. The steps are as follows: 2.1 Fragmentation of accessible chromatin regions: Genome fragmentation is performed using Tn5 transposase, where Tn5 is only coated with a 5' phosphate-modified linker sequence; 2.2 Oil-in-water generation: Cell barcoded gel beads are used to label fragmented DNA sequences within the same cell nucleus. Note that the difference from Example 1 is that the cell barcode tag has a special methylation modification that can maintain its own sequence unchanged during base conversion, as disclosed in patent application CN202410806830.7; 2.3 EM-seq conversion: After enrichment of labeled DNA, the C in the genomic DNA was converted to U using the NEB EM-seq conversion kit; 2.4 Amplification and library construction: DNA methylation library was constructed based on Truseq Read1 SeqPrimer single primer amplification followed by Tn5 secondary shearing. Note that the DNA polymerase used in this step must be urea-tolerant.

[0052] Step 3: Library quality control and sequencing 3.1 The library was screened with AMPure XP magnetic beads (150-1500 bp fragments), analyzed using an Agilent TapeStation 4200 fragment analyzer, and quantified using Qubit quantification before sequencing on an Illumina NovaSeq 6000 or SurfSeq 5000, both using the PE150 sequencing strategy.

[0053] 3.2 Data Analysis: SeekOne tools were used to perform UMI correction, alignment, and quality control analysis such as fragment and CPG quantity. The test results are shown in Table 2. Table 2. Comparison of single-cell methylation data from PBMC treated with alcohol or xSDS

[0054] As shown in Table 2, the normal nucleus C2 treated with the xSDS method detected only 21,906 fragments out of 38,896 read pairs, with a saturation of 43.68%. In contrast, the median cell of the ethanol-treated T2 sample detected 32,845 fragments out of a smaller 34,962 read pairs, a 49.9% increase compared to C2, and a saturation of 6.06%. The median cell of the isopropanol-treated T3 sample detected 29,163 fragments out of 28,632 read pairs, with an even lower saturation of 4.7%. Further, as shown in Table 2, statistics on CpG site detection in the median cell show that ethanol treatment increased the sensitivity of the median cell by 74.9% compared to the xSDS method (44,072 vs 25,196), while also increasing the genome coverage by 2.5 times from 0.08% to 0.20%.

[0055] These data demonstrate the advantages and reliability of the method by using alcohol treatment to increase the accessibility of heterochromatin DNA in the cell nucleus, which can significantly increase the average number of DNA fragments detected in single cells and the genome coverage.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A method for increasing the accessibility of chromatin DNA in a cell, characterized in that The following steps are involved: S10, providing samples to be tested; S20, mixing the sample to be tested with an alcohol reagent to permeabilize cells and expose the heterochromatin region of the sample to be tested, to obtain a pretreated sample; S30, fragmenting the DNA of the pretreated sample to obtain fragmented genomic DNA, performing reverse transcription on the RNA in the pretreated sample to obtain cDNA products, labeling the fragmented genomic DNA and the cDNA products, and separating the labeled genomic DNA and the labeled cDNA products; S40, constructing a genomic library and a transcriptome library for the labeled genomic DNA fragments and the labeled cDNA products, respectively, and sequencing them to obtain single-cell genomic information and transcriptome information in the sample to be tested.

2. A method for increasing the accessibility of chromatin DNA in a cell according to claim 1, characterized in that: In step S20, the alcohol reagent includes at least one of methanol, ethanol and isopropanol; and / or, The volume concentration of the alcohol reagent is 50-100%.

3. The method for improving the accessibility of chromatin DNA in cells according to claim 1, characterized in that: In step S20: The mixing temperature is -80°C to 25°C; and / or, The mixing time is 0.08h~24h.

4. A method for increasing the accessibility of chromatin DNA in cells according to claim 1, characterized in that: In step S30 , the reagents used in the fragmentation process include transposase or endonuclease.

5. The method for increasing the accessibility of chromatin DNA in cells according to claim 1, wherein: In step S30, the step of labeling the fragmented genomic DNA includes: Provide labels; The tags are added to the fragmented DNA by polymerase extension and / or ligase ligation.

6. A method for increasing the accessibility of chromatin DNA in cells according to claim 5, characterized in that: The carrier of the tag is a polymer microbead or a nano DNA microsphere; and / or, The labels on the same carrier are the same, and the labels on different carriers are different.

7. The method for increasing the accessibility of chromatin DNA in cells according to claim 1, wherein: In step S30, the step of labeling the cDNA product includes: S301, performing a reverse transcription reaction on the RNA in the pretreated sample to obtain a cDNA product; S302 , providing a tag sequence, and connecting the tag sequence to the cDNA product through a reverse transcription reaction to label the cDNA product.

8. The method for increasing the accessibility of chromatin DNA in cells according to claim 1, characterized in that: In step S40, the separation method includes nucleic acid affinity purification, antibody or streptavidin enrichment or PCR amplification enrichment.

9. A sequencing library, characterized in that The sequencing library includes a sequencing library constructed by the method for improving the accessibility of chromatin DNA in a cell according to any one of claims 1 to 8.

10. Use of a sequencing library constructed by the method for improving chromatin DNA accessibility in cells according to any one of claims 1 to 8 in high-throughput single-cell genome epigenetic modification detection.

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