Method for increasing chromatin DNA accessibility in cells and uses thereof

Abstract

The application discloses a method for improving chromatin DNA accessibility in cells and application thereof, relates to the technical field of gene sequencing and tissue cell sample analysis, and comprises the following steps: providing a sample to be detected; mixing the sample to be detected with an alcohol reagent to permeabilize cells and expose heterochromatin regions of the sample to be detected, so as to obtain a pretreated sample; performing fragmentation treatment on DNA of the pretreated sample, performing reverse transcription reaction on RNA in the pretreated sample, performing labeling on the fragmented DNA and the cDNA after the reverse transcription reaction, and separating labeled genomic DNA and cDNA; and performing library construction and sequencing on the fragments of the labeled genomic DNA and the cDNA product respectively, so as to obtain single-cell genomic information and transcriptome information of the sample to be detected. The alcohol reagent can open the heterochromatin without bias to obtain more nucleic acid fragments, improve the sensitivity of single-cell genome detection, does not affect RNA detection, and realizes high-throughput double-omics analysis of single-cell whole-genome DNA and RNA.

Description

TECHNICAL FIELD

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

[0002] High-throughput single-cell sequencing technology can realize parallel detection of molecular characteristics of thousands of cells, and has important value in analyzing cell heterogeneity. Its detection range covers multiple dimensions such as transcriptome, chromatin accessibility, copy number variation (CNV), somatic mutation and DNA methylation. At present, single-cell RNA sequencing (scRNA-seq) has gradually become a routine analysis method due to its mature technology, but single-cell genome analysis has not yet been widely applied. This technical bottleneck is mainly due to two key factors: first, each cell contains only double copies of genomic DNA; second, most of the genomic DNA exists in the form of highly compressed heterochromatin. These characteristics make it a great challenge to efficiently label single-cell DNA, resulting in generally low genome coverage. For example, when using Tn5 transposase to directly process human peripheral blood mononuclear cells (PBMCs) for single-cell chromatin accessibility sequencing (scATAC-seq), an average of only 10,000-20,000 DNA fragments can be obtained from a single cell, accounting for only about 0.1% of the whole genome.

[0003] At present, in order to improve genome coverage and ensure that homologous DNA has consistent cell labels, researchers have developed a variety of innovative solutions. The Tapestri platform of Mission Bio company is based on microfluidic technology, and uses a two-step water-in-oil-in-oil system to achieve cell capture and labeling: the first step of water-in-oil encapsulation encapsulates cells and proteases in microdroplets, allowing genomic DNA to be fully released; the second step of water-in-oil completes the cell-specific labeling (barcoding) of DNA. Similarly, the single-cell DNA copy number detection technology of 10X Genomics also uses a two-step water-in-oil strategy, but the first step of encapsulation is to fix DNA on water gel microbeads instead of microdroplets. Although this method can achieve DNA labeling at the single-cell level, it still has limitations such as high reagent cost and time-consuming operation. Taking a single cell as a natural reaction container, by improving the cutting efficiency of transposase through other methods, high-throughput single-cell DNA methylation detection has been successfully achieved. However, this method is subject to complex cell pretreatment procedures, the risk of DNA cross-contamination between cells, and the limited genome coverage due to insufficient sensitivity. These technical challenges highlight the need for further methodological breakthroughs in the field of single-cell genome analysis. SUMMARY

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

[0005] To achieve the above-mentioned purpose, the present application provides a method for improving the accessibility of chromatin DNA in cells, comprising the following steps:

[0006] S10, providing a sample to be tested;

[0007] S20, mixing the sample to be tested with an alcohol reagent to permeabilize the cells and expose the heterochromatin region of the sample to be tested, obtaining a pretreated sample;

[0008] S30, performing fragmentation treatment on the DNA of the pretreated sample to obtain fragmented DNA, performing reverse transcription reaction on the RNA in the pretreated sample to obtain cDNA product, and labeling the fragmented genomic DNA and the cDNA product;

[0009] S40, constructing a genomic library and a transcriptome library from the labeled genomic DNA fragments and the labeled cDNA product respectively and sequencing to obtain single-cell genomic information and transcriptome information of the sample to be tested.

[0010] In an embodiment, in step S20, the alcohol reagent includes at least one of methanol, ethanol and isopropanol.

[0011] In an embodiment, the volume concentration of the alcohol reagent is 50-100%; and / or,

[0012] The permeabilization treatment temperature is -80℃-25℃; and / or,

[0013] In an embodiment, the permeabilization treatment time is 0.08h-24h.

[0014] In step S30, the reagent used in the fragmentation treatment includes transposase or endonuclease.

[0015] In an embodiment, in step S30, the step of labeling the genomic DNA includes:

[0016] Providing a tag;

[0017] The tag is added to the fragmented DNA by polymerase extension and / or ligase ligation.

[0018] In an embodiment, the carrier of the tag is a polymeric microbead or a nano-DNA microsphere; and / or,

[0019] The tags on the same carrier are the same, and the tags on different carriers are different.

[0020] In an embodiment, in step S30, the step of labeling the cDNA product comprises:

[0021] S301, performing a reverse transcription reaction on the RNA in the pretreated sample to obtain a cDNA product;

[0022] S302, providing a tag sequence, and connecting the tag sequence to the cDNA product through a reverse transcription reaction to label the cDNA product.

[0023] In an embodiment, in step S40, the separation method comprises a nucleic acid affinity purification method, an antibody or streptavidin enrichment method, or a PCR amplification enrichment method.

[0024] The application also provides a sequencing library comprising the sequencing library constructed by the method for improving chromatin DNA accessibility in a cell according to any one of the above.

[0025] The application also provides the use of the sequencing library constructed by the method for improving chromatin DNA accessibility in a cell according to any one of the above in high-throughput single-cell genomic epigenetic modification detection.

[0026] The application can destroy the nuclear membrane and the higher-order structure of chromatin, fully expose the heterochromatin region, and thus avoid the damage to the sample caused by the traditional fixing, termination, and permeabilization, etc. complex steps, not only simplifying the experimental process, reducing the operation time and human error, but also maximizing the preservation of cell morphology integrity and intracellular nucleic acid on the basis of ensuring cell integrity. The alcohol reagent has 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 preserved, thereby improving the sensitivity and coverage of high-throughput single-cell genomic detection. In addition, the alcohol treatment does not affect the detection of RNA, enabling the simultaneous detection of DNA and RNA in high-throughput dual-omics at the single-cell level, thereby realizing the high-throughput co-detection of DNA and RNA in the same cell. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from the structures shown in these drawings without creative labor.

[0028] Figure 1 A schematic diagram of K562 cell nuclear Tn5 fragmentation DNA fragment distribution quality control with or without methanol treatment in an embodiment of the present application;

[0029] Figure 2 A comparative schematic diagram of the quality control index of K562 high-throughput single cell nuclear DNA sequencing with or without methanol treatment in an embodiment of the present application, which is the fragment ratio (Fraction Fragments overlapping Peaks) of the transcription start site (TSS) and the overlapping peak;

[0030] Figure 3 A comparative schematic diagram of the quality control index of K562 high-throughput single cell nuclear DNA sequencing with or without methanol treatment in an embodiment of the present application.

[0031] The purposes, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0032] In order to make the purposes, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, they are all the conventional products that can be purchased in the market. In addition, the meaning of "and / or" in the whole text includes three parallel solutions. For example, "A and / or B" includes the A solution, or the B solution, or the solution of A and B satisfying at the same time. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that the ordinary skilled in the art can realize it. When the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is not within the scope of protection required by the present application. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without creative labor are within the scope of protection of the present application.

[0033] Currently, to improve genome coverage and ensure that homologous DNA has consistent cell labels, researchers have developed various innovative solutions. The Tapestri platform from Mission Bio is based on microfluidic technology and uses a two-step water-in-oil-in-oil system to achieve cell capture and labeling: the first step of water-in-oil encapsulation packages cells and proteases together in microdroplets, allowing genomic DNA to be fully released; the second step of water-in-oil completes the DNA cell-specific labeling (barcoding). Similarly, the single-cell DNA copy number detection technology from 10X Genomics also uses a two-step water-in-oil strategy, but the first step of encapsulation is to fix DNA in hydrogel microbeads rather than microdroplets. Although this method can achieve DNA labeling at the single-cell level, it still has limitations such as high reagent cost and time-consuming operation.

[0034] The sci-MET technology developed by Andrew C. Adey's team takes a different approach, using a single cell as a natural reaction container. By treating chromatin with sodium dodecyl sulfate (xSDS) or lithium diiodosalicylate (LAND), the efficiency of transposase cleavage is improved, and high-throughput single-cell DNA methylation detection is successfully achieved. However, this method is subject to complex cell pretreatment procedures, the risk of DNA cross-contamination between cells, and limited genome coverage due to insufficient sensitivity. These technical challenges highlight the need for further methodological breakthroughs in the field of single-cell genomic analysis.

[0035] In view of this, the present application provides a method for improving the accessibility of chromatin DNA in cells and its application, comprising the following steps:

[0036] S10, providing a sample to be tested;

[0037] S20, mixing the sample to be tested with an alcohol reagent to permeabilize the cells and expose the heterochromatin region of the sample to be tested, obtaining a pretreated sample;

[0038] S30, fragmenting the DNA of the pretreated sample to obtain fragmented DNA, performing reverse transcription on the RNA in the pretreated sample to obtain a cDNA product, and labeling the fragmented genomic DNA and the cDNA product;

[0039] S40, constructing a genomic library and a transcriptome library from the labeled genomic DNA fragments and the labeled cDNA product, respectively, and sequencing to obtain single-cell genomic information and transcriptome information in the sample to be tested.

[0040] In the present application, by mixing the alcohol reagent with the sample to be tested, the nuclear membrane and the high-order structure of the chromatin of the sample to be tested can be destroyed, and the heterochromatin region of the sample to be tested can be fully exposed. The damage to the sample to be tested caused by the complex fixing, termination and permeation steps in the prior art is avoided. This not only simplifies the experimental process, but also reduces the operation time and potential human error. The alcohol reagent has the functions of removing nucleosomes, permeation and dehydration, and can maximize the retention of nucleic acids in cells while ensuring cell integrity, and also maintains the integrity of cell morphology. In addition, the pretreated sample is subjected to fragmentation processing. Since the alcohol treatment can open the heterochromatin without bias, the cutting efficiency in the fragmentation process is significantly improved, so that more nucleic acid fragments can be obtained, the nucleic acid information is retained, and the sensitivity and coverage of high-throughput single-cell genome detection are improved. Further, the treatment of the alcohol reagent does not affect the detection of RNA, and the traditional method cannot realize the co-detection of single-cell whole-genome DNA and RNA, so as to realize the co-detection of DNA and RNA of the same cell in high throughput.

[0041] It should be noted that compared with the traditional ATAC-seq (which relies on physiologically open chromatin), the present application can achieve random cutting of the whole genome by physical destruction, avoid the dependence on transcription start sites (TSS), and be suitable for cell types rich in heterochromatin (such as neurons and tumor stem cells). The present application breaks through the limitation of the traditional method that cannot realize the co-detection of single-cell whole-genome DNA and RNA.

[0042] In some embodiments, in step S20, the alcohol reagent includes at least one of methanol, ethanol and isopropanol. The alcohol reagent has the functions of removing nucleosomes, permeation and dehydration, and can maximize the retention of nucleic acids in cells while ensuring cell integrity, and also maintains the integrity of cell morphology.

[0043] 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 is further preferably 70-100%, such as 75% of methanol. This means that 75 mL of methanol is added to a 100 mL graduated cylinder. In the above concentration range, the alcohol reagent can fully expose and open the chromatin structure in the nucleus of the sample to be processed, so that more nucleic acid fragments can be provided subsequently, and the sensitivity of sequencing is improved.

[0044] In some embodiments, in step S20, the temperature of the mixing is -80°C to 25°C; any temperature in the above range can be used, in which the sample can be processed in a low-temperature environment, which can effectively reduce the enzyme activity, thereby preventing the degradation of DNA, RNA and protein, and also helps to maintain the original methylation pattern, avoid false positive or false negative results caused by temperature changes, and better protect the sample integrity and simplify the experimental process.

[0045] In some embodiments, the mixing time is 0.08h to 24h, in which the sample to be tested and the alcohol reagent are mixed to allow sufficient reaction.

[0046] In some embodiments, in step S30, the reagent used in the nucleic acid fragmentation process includes transposase or endonuclease treatment. In the process of using the alcohol reagent, the Tn5 transposase can fragment the DNA in the non-open region of the genome in the nucleus without bias, while keeping the morphology of the nucleus intact, thereby greatly increasing the detection amount of single-cell genomic fragments. Specifically, the transposase includes Tn5 transposase, and the endonuclease includes MboI.

[0047] Further, in step S30, the step of labeling the fragmented genomic DNA includes:

[0048] Providing a tag;

[0049] The tag is added to the fragmented DNA by polymerase extension and / or ligase ligation. The tag refers to a short sequence (DNA or RNA fragment) used to uniquely identify each sample or cell, which is designed to ensure that even if all samples are mixed together for processing, the source of each molecule or cell can still be traced.

[0050] In some embodiments, the carrier of the tag is a polymer microbead or a nano-DNA microsphere. The use of a tag carrier helps to 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 in complex samples.

[0051] In some embodiments, the tags on the same carrier are the same, and the tags on different carriers are different.

[0052] It should be noted that different tags are labeled on nucleic acid markers from different cell sources, the same or specific associated tags are labeled on nucleic acid markers from the same cell source; DNA nucleic acid molecules and RNA nucleic acid molecules from the same cell can be labeled with the same tag or a specific associated tag, so that they can be distinguished.

[0053] In an embodiment, in step S30, the step of labeling the cDNA product comprises:

[0054] S301, performing a reverse transcription reaction on the RNA in the pretreated sample to obtain a cDNA product;

[0055] S302, providing a tag sequence, and connecting the tag sequence to the cDNA product through a reverse transcription reaction to label the cDNA product.

[0056] It can be understood that, in order to prevent RNA degradation and ensure that RNA information can be accurately captured in subsequent processing, an alcohol reagent can be used as a protective agent. Alcohol can fix the intracellular components without damaging the integrity of the RNA, thereby providing stability for the subsequent separation steps. In addition, the tag sequence can be selected to have a cell barcode reverse transcription primer, which adds a tag sequence through reverse transcription reaction, or can not have a cell barcode, which adds a tag sequence on the cDNA product through a ligation reaction.

[0057] In some embodiments, in step S40, the separation method comprises nucleic acid affinity purification, antibody or streptavidin enrichment, or PCR amplification enrichment.

[0058] It can be understood that nucleic acid affinity purification is a technology for purifying nucleic acids based on the specific interaction between nucleic acids and specific ligands. For example, probes (such as oligonucleotides) that can specifically bind to target nucleic acid sequences can be designed, and then these probes can be used to separate target nucleic acids from complex mixtures. This method can highly specifically capture nucleic acid fragments of interest.

[0059] Antibody enrichment: commonly used in protein research, but can also be applied to nucleic acids labeled with specific antigen epitopes. By using antibodies against the epitope, the target molecules can be selectively enriched from complex mixtures.

[0060] Streptavidin enrichment method: based on the extremely high affinity between biotin-streptavidin. If the target nucleic acid or its tag is labeled with biotin, it can be enriched by streptavidin (or avidin) coated magnetic beads and other media. This is very common in genomics research, especially when constructing sequencing libraries to immobilize and purify DNA fragments with specific adapters.

[0061] PCR amplification enrichment method 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, thereby achieving effective enrichment. This method is not limited to genomic DNA, but also applies to cDNA (reverse transcribed from RNA), so it is also important in transcriptome analysis.

[0062] In some embodiments, after the step of separating the plurality of labeled nucleic acid fragments in step S40, the method further comprises 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 method of the present application, due to the improved sensitivity and coverage, a single cell DNA base modification detection library can be constructed through biochemical reactions, such as a DNA-5mC methylation and 5hmC hydroxymethylation detection library, so that high-throughput single cell genomic epigenetic modification detection can be realized subsequently.

[0063] The present application also provides a sequencing library comprising the sequencing library constructed by the method for improving the accessibility of chromatin DNA in cells according to any one of the above embodiments, which has the method for improving the accessibility of chromatin DNA in cells and all technical solutions of the method for improving the accessibility of chromatin DNA in cells, thus having the beneficial effects of the method for improving the accessibility of chromatin DNA in cells and the method for improving the accessibility of chromatin DNA in cells. The present application will not be repeated here.

[0064] It should be noted that sequencing can be selected from illumina, Huada or TrueSeq sequencer and all sequencing platforms compatible with the sequencing library of the present application. Those skilled in the art can modify the sequencing primer sequence in the sequencing library according to the requirements of different sequencing platforms to adapt to different sequencing platforms. These modifications do not affect the technical protection scope of the present patent.

[0065] The present application also provides a sequencing library constructed by the method for improving the accessibility of chromatin DNA in cells as described above for high-throughput single cell genomic epigenetic modification detection, such as a library construction kit.

[0066] The raw materials for the following examples are as follows:

[0067] K562 cell line: purchased from ATCC, ATCC number CRL-3344;

[0068] Human PBMC cells: purchased from AllCells, item number PB003F-C;

[0069] Commercialized SeekOne® DD single cell ATAC+RNA dual-omics kit: purchased from Beijing Xunyin Biotechnology Co., Ltd., item number K02901.

[0070] Example 1: Application of high-throughput single-cell genomic DNA and RNA co-detection

[0071] Step 1: Preparation of cell nucleus suspension and treatment with alcohol reagent

[0072] 1.1 Take fresh K562 cells, add NIB nuclear extraction buffer and treat on ice for 5 min, centrifuge at 500g and resuspend in PBS buffer, adjust the cell density to 1x10 6 mL, as shown, labeled as control sample C1; Figure 1

[0073] 1.2. Take 100 mL sample C1 and add dropwise to 500 mL pre-cooled methanol at -20°C, stand at -20°C for 30 min, centrifuge at 1000g to remove methanol and resuspend in PBS buffer, as shown, labeled as experimental sample T1; Figure 1

[0074] Step 2: Prepare DNA and RNA libraries from cell nucleus samples C1 and T1 according to the operation of the commercialized SeekOne® DD single cell ATAC+RNA dual-omics kit, brief steps as follows:

[0075] 2.1 Chromatin accessible region fragmentation: genomic fragmentation by Tn5 transposase;

[0076] 2.2 Water-in-oil generation: label chromatin DNA accessible region sequences and RNA sequences in the same cell nucleus by Barcode Beads, RNA based on semi-random primer reverse transcription labeling, ATAC sequence based on ligation strategy labeling;

[0077] 2.3 Pre-amplification: enrich the labeled products (RNA sequences and DNA sequences);

[0078] 2.4 Amplification and library construction: DNA library is directly amplified based on Truseq Read1 SeqPrimer and Nextera Read2SeqPrimer primers, and RNA library is amplified based on Truseq Read1 SeqPrimer and template switching primer, then the connection is broken to construct the library.​​

[0079] Step 3: Library quality control and sequencing

[0080] 3.1 The library was screened by AMPure XP magnetic beads (150-800 bp fragments), analyzed by Agilent TapeStation 4200 fragment analyzer and quantified by Qubit, and then sequenced by Illumina NovaSeq 6000 or SurfSeq5000, both using PE150 sequencing strategy. The test results are shown in Figure 1 , the C1 library without methanol treatment presents a peak pattern with nucleosome fragment size (140 bp) superimposed, which is a normal ATAC library; while the T1 nuclear library treated with methanol has a uniform fragment distribution, indicating that the chromatin structure in the nucleus is opened.

[0081] 3.2 Data analysis: UMI correction, alignment and TSS quality control analysis were performed using SeekArc tools, and the analysis results are shown in Figure 2 Figure 2 , A in Figure 2 is the TSS value of K562 nuclear C1 without methanol treatment, which is 7.47, while B in Figure 2 is the TSS value of methanol-treated nuclear T1, which is only 1.54; in addition, according to the Fraction Fragments overlapping Peaks index, Figure 2 C in Figure 2 is the index of K562 nuclear C1 without methanol treatment, which is 60.4%, while D in is the index of T1 treated with methanol, which is only 1.9%; these two indexes prove that the use of methanol reagent destroys the chromatin structure, and the DNA that can be broken by Tn5 is no longer limited to the transposition initiation site (TSS) under physiological conditions, but is randomly broken on the genome, so there is no Peak peak.

[0082] Figure 3 In addition, the number of DNA fragments detected by the median cell was further analyzed, and the test results are shown in , the median cell of T1 sample treated with methanol detected 75017 fragments under the condition of 82570 read pairs; while the normal nucleus C1 without methanol treatment only detected 12367 fragments under the condition of 104074 read pairs, with a difference of 6 times, which proves that the use of alcohol treatment can significantly increase the average detection of DNA fragments in single cells after improving the accessibility of heterochromatin DNA in the nucleus.

[0083] In addition, as shown in Table 1, the analysis of the RNA library proves that the method of treating the nucleus with methanol does not affect the detection of RNA.

[0084] Table 1: Comparison of K562 high-throughput single-cell nucleus RNA sequencing core quality control indicators with or without methanol treatment

[0085] Example 2: Application of high-throughput single-cell methylation detection using alcohol pretreatment

[0086] Step 1: Preparation and differential treatment of PBMC cell nucleus suspension

[0087] 1.1 Take human PBMC cells, treat with 1x lysis buffer on ice for 3 min, centrifuge at 500g, resuspend in PBS buffer, and adjust the cell density to 1x10 6 mL;

[0088] 1.2 Take 100 mL of nucleus suspension and add 500 mL of -20°C pre-cooled 100% ethanol dropwise, mark as experimental sample T2, and take another 100 mL of nucleus suspension and add 500 mL of -20°C pre-cooled 100% isopropanol dropwise, mark as experimental sample T3, both T2 and T3 are placed at -20°C for 30 minutes, centrifuge at 1000g to remove ethanol or isopropanol, and resuspend in PBS buffer; take another 100 mL of nucleus suspension and treat the nucleus according to the xSDS protocol in sci-MET technology, i.e. first fix the nucleus with 1.5% formaldehyde and then remove the nucleosomes in the nucleus with 0.3% SDS, mark as control sample C2;

[0089] Step 2: Prepare single-cell methylation libraries from nucleus samples C2, T2 and T3 according to the operation of commercial SeekOne® DD single-cell methylation detection kit, brief steps as follows:

[0090] 2.1 Chromatin accessible region fragmentation: fragment the genome by Tn5 transposase, where the Tn5 is only coated with a 5' phosphate-modified linker sequence;

[0091] 2.2 Water-in-oil generation: label the fragmented DNA sequences in the same nucleus with Cell Barcoded Gel Beads; note that the difference from Example 1 is that the cell barcode label has a special methylation modification that can remain unchanged during base conversion, as disclosed in patent application CN202410806830.7;

[0092] 2.3 EM-seq conversion: after enrichment of the labeled DNA, use the NEB EM-seq conversion kit to convert C to U in the genomic DNA;

[0093] 2.4 Amplification and library construction: DNA methylation library is constructed based on the Truseq Read1 SeqPrimer single primer amplification and Tn5 secondary breakage. Note that the DNA polymerase used in this step must be resistant to uracil bases.

[0094] Step 3: Library quality control and sequencing

[0095] 3.1 The library is screened by AMPure XP magnetic beads (150-1500 bp fragments), analyzed by Agilent TapeStation4200 fragment analyzer and quantified by Qubit, and then sequenced by Illumina NovaSeq 6000 or SurfSeq5000, both using PE150 sequencing strategy.

[0096] 3.2 Data analysis: UMI correction, alignment, and fragments and CPG quantity quality control analysis are performed using SeekOne tools. The test results are shown in Table 2.

[0097] Table 2. Comparison of single-cell methylation data indicators detected by using alcohol treatment or xSDS method to treat PBMC

[0098]

[0099] From Table 2, it can be seen that the normal cell nucleus C2 treated by xSDS method measured only 21906 fragments in 38896 read pairs, with a saturation degree of 43.68%; while the median cell of T2 sample treated by ethanol measured 32845 fragments in 34962 read pairs, which increased by 49.9% compared with C2, and the saturation degree reached 6.06%; the median cell of T3 sample treated by isopropanol measured 29163 fragments in 28632 read pairs, and the saturation degree was also lower, which was 4.7%; further from Table 2, it can be seen that the statistics of CpG sites detected in the median cell showed that the sensitivity of ethanol treatment was increased by 74.9% (44072 vs 25196) compared with xSDS method, and the genome coverage was also increased from 0.08% to 0.20%, which was 2.5 times.

[0100] These data prove that the use of alcohol treatment to improve the accessibility of heterochromatin DNA in the nucleus can significantly increase the average number of DNA fragments detected in single cells and the genome coverage, and the method is reliable.

[0101] The above merely describes the preferred embodiments of the present application, and is not intended to limit the patent scope of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the patent protection scope of the present application.

Claims

1. A method for improving the accessibility of chromatin DNA in cells, said method for non-disease diagnosis or non-treatment purposes, characterized in that, The method includes the following steps: S10. Provide a sample to be tested, wherein the sample to be tested is an unlysaturated sample that has not been mixed with aldehydes for fixation; S20. The sample to be tested is mixed with an alcohol reagent to fix, permeate and expose all heterochromatin regions of the sample in one step, to obtain a pretreated sample. The alcohol reagent includes at least one of methanol, ethanol and isopropanol. S30. The DNA of the pretreated sample is fragmented to obtain fragmented genomic DNA. The RNA in the pretreated sample is reverse transcribed to obtain cDNA product. The fragmented genomic DNA and the cDNA product are labeled, and the labeled genomic DNA and the labeled cDNA product are separated. S40. The labeled genomic DNA fragments and labeled cDNA products are used to construct genomic libraries and transcriptome libraries, respectively, and then sequenced to obtain single-cell genomic and transcriptome information in the sample to be tested.

2. The method for improving chromatin DNA accessibility in cells as described in claim 1, characterized in that, In step S20, the volume concentration of the alcohol reagent is 50-100%.

3. The method for improving chromatin DNA accessibility in cells as described in claim 1, characterized in that, In step S20: The mixing temperature is -80℃ to 25℃; and / or, The mixing time is 0.08h to 24h.

4. The method for improving chromatin DNA accessibility in cells as described in claim 1, characterized in that, In step S30, the reagents used for the fragmentation process include transposases or endonucleases.

5. The method for improving chromatin DNA accessibility in cells as described in claim 1, characterized in that, In step S30, the step of tagging the fragmented genomic DNA includes: Provide tags; The tag is added to the fragmented DNA using polymerase extension and / or ligase ligation.

6. The method for improving chromatin DNA accessibility in cells as described in claim 5, characterized in that, The carrier of the tag is a polymer microbead or a DNA nanosphere; and / or, The labels on the same carrier are the same, and the labels on different carriers are different.

7. The method for improving chromatin DNA accessibility in cells as described in claim 1, characterized in that, In step S30, the step of labeling the cDNA product includes: S301. Perform reverse transcription on the RNA in the pretreated sample to obtain cDNA product; S302. Provide a tag sequence and tether the tag sequence to the cDNA product via reverse transcription to label it.

8. The method for improving chromatin DNA accessibility in cells as described in 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 using the method for improving chromatin DNA accessibility in cells as described in any one of claims 1 to 8.

10. The application of a sequencing library constructed using the method for improving chromatin DNA accessibility in cells as described in any one of claims 1 to 8 in high-throughput single-cell genome epigenetic modification detection, wherein the application is for non-disease diagnosis or non-therapeutic purposes.

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