Methods for constructing sequencing libraries for detecting single-cell dna methylation and chromatin structure

Abstract

The application discloses a method for constructing a sequencing library for detecting single-cell DNA methylation and chromatin structure in the field of nucleic acid determination. The technical problem to be solved by the application is how to efficiently, quickly and at low cost capture single-cell DNA methylation and single-cell chromatin structure. The application uses DNA ligase to connect adjacent chromatin fragments after end repair after cross-linking and using micrococcus nuclease for genome fragmentation of dissociated single cells; then each single cell nucleus is labeled with a unique barcode combination tag; then all the obtained single cell nuclei are de-cross-linked and DNA purified to obtain target DNA fragments; the target DNA fragments are subjected to methylation sequencing library construction to obtain base-converted target DNA fragments, and a sequencing library is obtained. The application can be used for developing or preparing a product for cell subpopulation or screening of abnormally developed cells.

Description

Technical Field

[0001] This invention relates to a method for constructing sequencing libraries for detecting DNA methylation and chromatin structure in single cells in the field of nucleic acid assays. Background Technology

[0002] Epigenetics refers to modifications that alter gene expression and phenotype without changing the DNA sequence. It plays a crucial role in regulating complex biological processes, including fertilization, early embryonic development, and aging. In eukaryotic cells, these mechanisms involve multiple levels, including covalent modifications of DNA molecules such as DNA methylation, and chromosome modifications such as histone modifications and chromatin accessibility. In addition to these epigenetic modifications, chromosome conformation—the way chromosome fibers fold within the cell nucleus—also plays an important role in these biological processes.

[0003] DNA methylation is a fundamental epigenetic modification, primarily involving the addition of a methyl group to the fifth carbon of the cytosine ring to form 5-methylcytosine. In mammals, DNA methylation mainly occurs at CpG dinucleotides, which often aggregate to form regions called CpG islands. CpG islands are typically hypomethylated and are located in approximately 70% of gene promoter regions. Hypomethylation in promoter regions is associated with active transcription, while gene bodies and intergenic regions are usually hypermethylated. The dynamic changes in DNA methylation are mediated by reversible enzymatic reactions, switching between methylation and demethylation. The establishment and maintenance of DNA methylation are mediated by the DNA methyltransferase family, while the reversibility of DNA methylation is achieved by the active demethylation process mediated by TET enzymes. TET enzymes can oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine, some of which serve as intermediates, enabling cells to respond to developmental and environmental signals and fine-tune gene expression and chromosome state.

[0004] Chromosomal conformation is also a key aspect of epigenetic regulation, forming a multi-layered three-dimensional structure within the eukaryotic cell nucleus. This hierarchical structure begins with the smallest unit, the nucleosome, and gradually evolves into larger structures, including chromatin loops, topologically associated domains (TADs), chromosomal compartments, and the largest structural unit, the chromosome domain. Chromosomal conformation is highly dynamic and regulated by chromatin complexes and structural proteins. For example, the ATP-dependent chromatin remodeling complex is responsible for the repositioning of nucleosomes to regulate chromosome conformation. Chromosomal structural proteins such as CTCF and cohesin mediate the loop extrusion mechanism, in which the cohesin complex extrudes and stops at the CTCF binding site, thereby forming chromatin loops and TAD boundaries. The hierarchical and dynamic changes in chromosome structure help ensure the precision and flexibility of gene regulation.

[0005] One of the most important discoveries of the Human Genome Project was that the human genome is largely composed of non-coding regions containing numerous cis-regulatory elements that are crucial for spatiotemporal gene expression in specific cell types of multicellular organisms. With the development of sequencing technologies, especially single-cell sequencing, extensive cellular heterogeneity within complex tissues and comprehensive epigenetic mechanisms in gene regulation, including DNA methylation and chromosome organization, have been revealed.

[0006] Chromosome organization sequencing technology primarily relies on DNA proximity junctions to identify chromosome contacts. In 2009, Hi-C technology, by improving sequencing library efficiency through a biotin-pulp step, revealed a map of chromosome interactions across the entire mammalian genome. Subsequently, this principle was further applied to the single-cell level by isolating individual cell nuclei and performing indexed PCR amplification. In the following years, various single-cell chromosome organization sequencing methods were developed, revealing dynamic and heterogeneous chromosome organization structures widely present in different cells.

[0007] DNA methylation sequencing methods rely on converting methylated or unmethylated cytosine into different base classes. Currently, there are two main strategies for cytosine conversion: sulfite conversion and enzymatic conversion. Sulfite conversion is considered the gold standard for DNA methylation detection due to its precise methylation quantification, but it suffers from significant DNA degradation during the conversion process. In recent years, enzymatic conversion methods such as EM-seq and TET-assisted pyridineborane sequencing have been developed to reduce DNA damage and accommodate low DNA input volumes. The first single-cell DNA methylation sequencing method was single-cell simplified representative bisulfite sequencing, which covered more than one million CpG sites per cell and revealed a fine demethylation map of mouse post-fertilized embryos. Subsequently, single-cell whole-genome DNA methylation analysis methods based on sulfite conversion were developed, capable of covering approximately 20% of the CpG sites in the mouse genome. To improve cell throughput, single-cell DNA methylation analysis methods based on combinatorial indexes or droplet platforms have also been developed, enabling high-throughput analysis of thousands of cells.

[0008] With the development of single-cell technology, single-cell multi-omics technology has emerged, capable of simultaneously capturing two or more modalities within a single cell, thus providing a more comprehensive understanding of cellular heterogeneity and multimodal integration. Existing methods that can simultaneously analyze DNA methylation and chromosome architecture in the same single cell provide a pathway to study their interactions at the single-cell level. In short, these methods perform sulfite conversion on neighboring linkage products derived from 3C or Hi-C at the single-cell level, thereby simultaneously capturing information on DNA methylation and chromosome interactions.

[0009] DNA methylation and chromosome interaction information have important applications in tracking cell differentiation pathways and constructing epigenetic lineages. For example, for nerve cells, after capturing epigenetic signals such as DNA methylation and chromosome interactions, the neuronal developmental trajectory can be calculated, thereby constructing an epigenetic reference assessment system for the degree of neural differentiation and developmental maturity. Using this epigenetic reference system, the neural development of different samples can be comprehensively evaluated and scored, thus enabling the evaluation of the sample's neural developmental status and the screening of abnormal deviations. Furthermore, epigenetic signals can be used to find mutation sites in neurons of neurodegenerative diseases and neurodevelopmental disorders that are difficult to detect using traditional genetics, thereby better facilitating genetic screening and the development of related biomarkers. In summary, various epigenetic signals such as DNA methylation and chromosome interactions have broad application prospects in sample quality control and abnormal state screening.

[0010] However, existing methods for co-capturing single-cell DNA methylation and chromatin structure still have significant shortcomings: 1) Existing methods are all low-throughput methods, insufficient in cell capture, with only a few hundred cells captured per experiment, limiting their use in tissues with high heterogeneity and large cell counts; 2) Existing methods are time-consuming, requiring several days for a single experiment, and large-scale library construction often requires a large amount of time and materials; 3) Existing methods are expensive, which is detrimental to large-scale use and hinders widespread adoption. These problems limit research progress and clinical application in the field of single-cell DNA methylation and chromatin structure. Summary of the Invention

[0011] The technical problem to be solved by this invention is how to construct a high-throughput, rapid, and low-cost co-capture library of single-cell DNA methylation and single-cell chromatin structure.

[0012] To address the aforementioned technical problems, this invention first provides a method for constructing a sequencing library for detecting single-cell DNA methylation and chromatin structure, the method comprising the following steps: The method involves cross-linking, genome fragmentation, and chromatin end repair of dissociated single cells or single cell nuclei to obtain chromatin end-repaired single cell nuclei or single cells 1, characterized in that: the genome fragmentation is performed using micrococcal nuclease; the method further includes: 1) Use DNA ligase to perform chromatin fragment ligation on the single cell nucleus or single cell 1 to obtain single cell nucleus or single cell 2; 2) Each single cell nucleus or single cell 2 is labeled with different barcode combinations to distinguish the DNA from different single cell nuclei or single cells, resulting in single cell nuclei or single cells 3; 3) Decrosslink all single-cell nuclei or single-cell cells and collect DNA fragments; 4) Construct a methylation sequencing library from the DNA fragment to obtain the base-converted fragment, and obtain a sequencing library for detecting DNA methylation and chromatin structure in single cells.

[0013] In some embodiments, the method does not use transposases. The chromatin fragment end ligation of the single-cell nucleus or single-cell I using DNA ligase does not include ligating sequencing adapters.

[0014] Chromatin fragment proximity ligation involves joining spatially close DNA fragments into a new DNA molecule. In some embodiments, this is achieved using DNA ligases.

[0015] The method does not include the step of removing nucleosomes. Step 3) The collected DNA fragments include DNA in nucleosomes and DNA that is not wrapped around histones (naked DNA).

[0016] In the above method, the genome fragmentation is performed using micrococcal nuclease, and the amount of micrococcal nuclease used can be 1500 U / million cell nuclei or cells; the cutting can be performed at 37°C for 10 minutes. The genome fragments obtained by the genome fragmentation (existing in the form of chromatin fragments) are 100bp-200bp in size, mainly around 120bp.

[0017] In one specific embodiment of the present invention, the steps of repairing the chromatin fragment ends of all the single cell nuclei or single cells 1 are to flatten the chromatin fragments and add phosphate groups.

[0018] In one embodiment, the methylation sequencing library construction is achieved using the Enzymatic Methyl-seq (EM-seq) library construction method, which may specifically include the base conversion process: modifying the target DNA fragment with cytosine and then deaminating the modified cytosine.

[0019] In one embodiment, the base conversion treatment of the target DNA fragment to obtain the base-converted target DNA fragment includes the following steps: modifying and protecting the cytosine in the target DNA fragment by converting both 5mC (methylated cytosine) and 5hmC (5-hydroxymethylcytosine) to 5caC (5-carboxycytosine) to obtain the cytosine-modified target DNA fragment; then using an APOBEC family deaminase (such as APOBEC3A) to perform an enzymatic deamination reaction on the unmodified unmethylated cytosine (C) of the cytosine-modified target DNA fragment, directly converting it to uracil (U).

[0020] In the above method, the different barcode combination labels can be implemented using a connection-based combination label method.

[0021] The ligation-based combinatorial barcoding method is a strategy for adding multiple combinatorial tags to nucleic acid molecules through a ligation reaction. This method involves multiple rounds of "pooling-barcoding-mixing" operations to assign a unique DNA barcode (a combination of n barcodes, where n is a natural number greater than or equal to 2, and each barcode in the n barcode combination can be different) to each chromatin fragment in a single cell nucleus or single cell. This allows for tracing the single-cell origin of each chromatin fragment even after mixed sequencing.

[0022] In the above method, the crosslinking agent may include formaldehyde and / or bis(succinimide) glutarate.

[0023] The above method may further include the step of adding sequencing adapters by performing PCR amplification on the target DNA fragment after base conversion using primers with sequencing platform adapter sequences.

[0024] In the above method, each of the different barcode combination tags is composed of barcode1, barcode2, and barcode3; barcode1 is a double-stranded DNA obtained by annealing any of the following single-stranded nucleotide pairs: single-stranded nucleotide pairs composed of sequences 1 and 7, sequences 2 and 8, sequences 3 and 9, sequences 4 and 10, sequences 5 and 11, and sequences 6 and 12; barcode2 is any of the 96 double-stranded DNAs obtained by annealing any one of the 96 single-stranded DNAs from sequences 13 to 108 with the single-stranded DNA shown in sequence 205; barcode3 is any of the 96 double-stranded DNAs obtained by annealing any one of the 96 single-stranded DNAs from sequences 109 to 204 with the single-stranded DNA shown in sequence 206.

[0025] In one specific embodiment of the present invention, the method described above further includes a step of permeabilizing all the single cell nuclei or single cells 1: treating the single cell nuclei or single cells with ionic detergent SDS and Triton X-100 to achieve permeabilization of the nuclear membrane and loosen chromatin, thereby improving the efficiency of barcode and enzyme, while controlling the concentration to ensure the maintenance of the single cell nucleus or single cell state.

[0026] To address the aforementioned technical problems, the present invention also provides a composition for constructing a sequencing library for detecting single-cell DNA methylation and chromatin structure, wherein the nucleic acid composition comprises the barcode combination tag and micrococcal nuclease described above.

[0027] The above composition may also include at least one of the following substances: DNA ligase, formaldehyde, SDS, proteinase K, TET2 enzyme, and deaminase.

[0028] To address the aforementioned technical problems, this invention also provides the application of the method in preparing products for detecting cell developmental trajectories, preparing products for screening abnormally developing cells, preparing cell clustering products, or preparing products for tracking cell differentiation pathways. The method described above is the same as the method described in the preceding text.

[0029] To address the aforementioned technical problems, this invention also provides the application of the composition in constructing sequencing libraries for detecting single-cell DNA methylation and chromatin structure. The composition is the one described above.

[0030] The applications or methods described above are not for disease diagnosis. They are not intended to directly obtain disease diagnoses or health status results from living humans or animals.

[0031] The above applications or methods are for non-disease treatment purposes. They are not intended to restore or restore health or reduce suffering in living human or animal bodies.

[0032] This invention provides a novel co-capture technology for single-cell DNA methylation and single-cell chromatin structure: 3M-seq.

[0033] Advantages compared to existing technologies: The experimental procedure of this invention achieves high-throughput cell capture; reduces the cost of constructing co-capture sequencing libraries for single-cell DNA methylation and single-cell chromatin structure; and reduces experimental time. This invention is applicable to a wider range of tissues and is conducive to large-scale deployment. Attached Figure Description

[0034] Figure 1 This is a flowchart illustrating the method of the present invention.

[0035] Figure 2 This figure compares the parameters of the method of the present invention with other single-cell three-dimensional genomics technologies, single-cell methylation technologies, and co-capture technologies for single-cell DNA methylation and single-cell chromatin structure. The figure above shows a comparison of the method of the present invention with existing single-cell methylation detection technologies (M1 represents scWGBS, M2 represents scRRBS, M3 represents snmC-seq, M4 represents sciMET, M5 represents sciMETv2, M6 represents scBS-seq, M7 represents scMspJI-seq, M8 represents Drop-BS, M9 represents sciEM) and two other existing co-capture technologies for single-cell DNA methylation and single-cell chromatin structure (C1 represents scMethyl-HiC, C2 represents sn-m3C-seq) in terms of cell throughput and capture efficiency of methylation signals per cell. Figure 2 The lower figure shows a comparison of the method of the present invention with existing single-cell three-dimensional genome detection technologies (H1 represents scHi-C, H2 represents sci-Hi-C, H3 represents snHi-C, H4 represents Dip-C, H5 represents s3-GCC, H6 represents scHi-C v2, H7 represents snHi-C, H8 represents Droplet Hi-C, H9 represents scMicro-C, H10 represents scNanoHi-C) and two other existing single-cell DNA methylation and single-cell chromatin structure co-capture technologies (C1 represents scMethyl-HiC, C2 represents sn-m3C-seq) in terms of cell throughput and chromatin structure interaction capture efficiency per cell. This method is the method of Example 1 (steps 1-11).

[0036] Figure 3 This is a parameter comparison chart showing the library preparation time and usage cost of the method of the present invention compared with existing single-cell DNA methylation and single-cell chromatin structure co-capture technologies. This method is the method of Example 1 (steps 1-11).

[0037] Figure 4 This image shows a comparison between the chromatin structure obtained by this invention and the results obtained by common chromatin structure capture methods. The left image shows the chromatin structure result at 500kb resolution, and the right image shows the chromatin structure result at 50kb resolution. This method is the method of Example 1 (steps 1-11).

[0038] Figure 5 This is a comparison chart of the DNA methylation results obtained by the present invention and the DNA methylation detection results of common DNA methylation capture methods. The upper figure shows the DNA methylation results obtained by the method of the present invention (i.e., the method of steps 1-11 in Example 1); the lower figure shows the DNA methylation detection results obtained by the method in the prior art.

[0039] Figure 6 The provided sequence is the single-stranded primer adapter sequence used to generate barcode 1. The nucleotide sequences of primers mR01_#01_RE to mR01_#06_RE correspond to sequences 1-6 in the sequence listing (the 5' terminal nucleotides of sequences 1-6 are all phosphorylated); the nucleotide sequences of primers mR01_#01_NRE to mR01_#06_NRE correspond to sequences 7-12 in the sequence listing; the fifth position of nucleotide C at position 19 in sequences 1-6 is methylated to 5-methyldeoxycytidine (i.e., i5MedC represents 5-methyldeoxycytidine 5-Me-dC).

[0040] Figure 7 The provided sequence is the single-stranded primer adapter sequence used to generate barcode2. The nucleotide sequences of primers R02#01-R02#96 correspond to sequences 13-108 in the sequence listing (the 5' terminal nucleotides of sequences 13-108 are all phosphorylated).

[0041] Figure 8 The provided sequence is the single-stranded primer adapter sequence used to generate barcode 3. The nucleotide sequences of primers mR03_#01-mR03_#96 correspond to sequences 109-204 in the sequence listing. In sequences 109-204, the fifth position of nucleotide C at position 19 is modified with a methyl group to become 5-methyldeoxycytidine (i.e., i5MedC represents 5-methyldeoxycytidine 5-Me-dC).

[0042] Figure 9Four P5 series primers (P5_5-P5_8) and four N7 series primers (N7_24-N7_27) are listed. Figure 9 The sequence of primer P5_5 corresponds to sequence 215 in the sequence listing, and the 70th nucleotide of sequence 215 is a T modified with a thiophosphate group; the sequence of primer P5_6 corresponds to sequence 216 in the sequence listing, and the 70th nucleotide of sequence 216 is a T modified with a thiophosphate group; the sequence of primer P5_7 corresponds to sequence 217 in the sequence listing, and the 70th nucleotide of sequence 217 is a T modified with a thiophosphate group; the sequence of primer P5_8 corresponds to sequence 218 in the sequence listing, and the 70th nucleotide of sequence 218 is a T modified with a thiophosphate group. Figure 9 The sequence of primer N7_24 corresponds to sequence 219 in the sequence listing; the sequence of primer N7_25 corresponds to sequence 220 in the sequence listing; the sequence of primer N7_26 corresponds to sequence 221 in the sequence listing; and the sequence of primer N7_27 corresponds to sequence 222 in the sequence listing. "*T" represents a T modified with a thiophosphate group.

[0043] Figure 10 To utilize the method of this invention to co-capture methylation and chromatin structure of cerebral cortex cells in human embryos at 23 weeks of gestation, a dimensionality-reduced clustering map of approximately 10,000 cells was obtained based on the capture results. The results showed that all cerebral cortex cell types at this stage were successfully identified, demonstrating the co-capture and clustering capability of the method of this invention for DNA methylation and chromatin structure in complex tissues.

[0044] Figure 11 To utilize the method of this invention to co-capture methylation and chromatin structure of cerebral cortex cells in human embryos at 23 weeks of gestation, developmental trajectories of excitatory and inhibitory neurons were obtained based on the capture results. The left image shows the developmental trajectory of excitatory neurons; the right image shows the developmental trajectory of inhibitory neurons. These trajectories, from early cell types such as neural progenitor cells to mature excitatory and inhibitory neurons, demonstrate the ability of the method of this invention to identify developmental maturity by extracting epigenetic signals from complex tissues. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] The solution composition and preparation method in this embodiment of the invention are as follows: 3M-seq buffer #1 stock solution (MB1 stock solution) (10 mL): 100 μL of NaCl (5 M, Sigma, S7653), 100 μL of Tris-HCl (pH=7.5, 1 M, Sigma, S7653), 50 μL of MgCl2 (1 M, Sigma, 63069), 10 μL of CaCl2 (1 M, Maclean's, C885126), and 9340 μL of sterile water were added to make up the total volume.

[0048] 3M-seq buffer #2 (MB2) (10 mL): Add 100 μL of NaCl (5 M), 100 μL of Tris-HCl (pH=7.5, 1 M), 100 μL of MgCl2 (1 M), and 9700 μL of sterile water to make up the total volume.

[0049] 3M-seq buffer #3 (MB3) (10 mL): 500 μL of Tris-HCl (pH=7.5, 1 M), 100 μL of MgCl2 (1 M), and 9400 μL of sterile water.

[0050] MNase dilution buffer (500 μL): Add 5 μL of NaCl (5 M), 5 μL of Tris-HCl (pH=7.5), 1 μL of EDTA (0.5 M, Invitrogen, AM9261), 250 μL of glycerol, and 239 μL of sterile water to make up the total.

[0051] MNase working solution stock solution of 200 U / μL: Add 100,000 U of micrococcal nuclease (MNase, NEB, MO247S) to 500 μL of MNase dilution buffer, mix well, and obtain a MNase working solution stock solution with a concentration of 200 U / μL. Aliquot the stock solution into 50 μL tubes and store at -20°C for later use.

[0052] Dissociation buffer (2 mL): 500 μL of sucrose (1 M, Sigma, S7903), 50 μL of KCl (1 M, Sigma, P9333), 40 μL of MgCl2 (250 mM), 20 μL of Tris-HCl (pH=8.0, 1 M), 20 μL of DTT (0.1 M, Sigma, D9779), 40 μL of a mixture of protease inhibitors (50×, Roche, 05056489001), and 1330 μL of sterile water were added to make up the total.

[0053] Crosslinking buffer (10 mL): 2500 μL of sucrose (1 M), 250 μL of KCl (1 M), 200 μL of MgCl2 (250 mM), 100 μL of DTT (0.1 M), and 6650 μL of sterile water were added to make up the total.

[0054] bis(succinimide) glutarate (DSG) solution (3 mM): Dissolve 1 mg DSG (Thermo Scientific, 2059) in 10 μL of DMSO to prepare a 300 mM stock solution, then add 990 μL of PBS and mix well to obtain a 3 mM DSG solution.

[0055] Micro-C Buffer #1 Working Solution (MB1 Working Solution, 300 μL): 286.25 μL of MB1 stock solution, 6.25 μL of protease inhibitor mixture (50×), and 7.5 μL of digitalis saponins (2%, Sigma, D141).

[0056] End-of-phase repair reaction solution (ECB, 45 μL): 5 μL of 10× NEBuffer 2.1 (NEB, B7202V), 10 μL of ATP (10 mM, NEB, R0441), 2.5 μL of DTT (0.1 M), 25 μL of sterile water, and 2.5 μL of T4 polynucleotide kinase (T4 PNK) (10 U / μL, NEB, M0201S).

[0057] End-lengthening reaction solution (ELB, 25 μL): 2.5 μL of 10× T4 DNA ligase buffer (NEB, M0202L), 1 μL of dNTP mixture (10 mM, Thermo Fisher Scientific), 0.125 μL of BSA (20 mg / mL), and 21.375 μL of sterile water to make up the total.

[0058] Ligation reaction solution (250 μL): 12.5 μL of T4 DNA ligase (400 U / µL, NEB, M0202L), 1.25 μL of BSA (20 mg / mL), 25 μL of 10× T4 DNA ligase buffer, and 211.25 μL of sterile water to make up the total.

[0059] The first round of barcode ligation reaction solution (BLB#1), 200 μL per reaction system: 20 μL of 10× T4 DNA ligase buffer, 1.25 μL of BSA, 10 μL of T4 DNA ligase, 25 μL of PEG-8000 (50%, Beyotime, R0056), 35 μL of barcode #1 adapter (100 μM, Sangon Biotech), and 108.75 μL of sterile water to make up the difference. The barcode #1 adapter was obtained through the following steps: 100 μL of six NREs (200 μM, mR01_#01_NRE to mR01_#06_NRE) single-stranded primers and 100 μL of six REs (200 μM, mR01_#01_RE to mR01_#06_RE) single-stranded primers were mixed and annealed to obtain the six barcode #1 adapters. Figure 6 Annealing mR01_#01_RE with mR01_#01_NRE yields double-stranded primer mR01_#01; annealing mR01_#02_RE with mR01_#02_NRE yields double-stranded primer mR01_#02; annealing mR01_#03_RE with mR01_#03_NRE yields double-stranded primer mR01_#03; annealing mR01_#04_RE with mR01_#04_NRE yields double-stranded primer mR01_#04; annealing mR01_#05_RE with mR01_#05_NRE yields double-stranded primer mR01_#05; and annealing mR01_#06_RE with mR01_#06_NRE yields double-stranded primer mR01_#06. Reaction conditions: 95°C for 5 minutes; cool to 4°C at a rate of 0.1°C per second; hold at 4°C.

[0060] The first round of barcode ligation replenishment solution (BLB#1 replenishment solution), 50 μL per reaction: 5 μL of 10× T4 DNA ligase buffer, 2.5 μL of T4 DNA ligase, and 42.5 μL of sterile water.

[0061] Second or third round barcode ligation master mix (BLB#2 or BLB#3), total volume 3000 μL: 400 μL 10× T4 DNA ligase buffer, 50 μL BSA, 2450 μL sterile water, 100 μL T4 DNA ligase.

[0062] The second round of barcode #2 adapters was obtained through the following steps: 6 μL of 96 different 100uMR02 single-stranded primer adapters (R02#01-R02#96) and 6 μL of linker R02 (sequence 205: 5'-CGAATGCTCTGGCCTCTCAAGCACGTGGAT-3') were mixed with 38 μL of water and annealed to obtain the 96 barcode #2 adapters. Figure 7 Annealing R02#01 with linkerR02 yields double-stranded primer R02#01-L2, annealing R02#02 with linkerR02 yields double-stranded primer R02#02-L2, ..., annealing R02#96 with linkerR02 yields double-stranded primer R02#96-L2. Reaction conditions: 95°C for 5 minutes; cooling to 4°C at a rate of 0.1°C per second; holding at 4°C.

[0063] The third round of barcode #3 adapters was obtained through the following steps: 6 μL of 96 different 100 μM mR03 single-stranded primer adapters (mR03_#01-mR03_#96) and 6 μL of linkerR03 (sequence 206: 5'-GGTCTGAGTTCGCACCGAAACATCGGCCAC-3') were mixed with 38 μL of water and annealed to obtain the 96 barcode #3 adapters. Figure 8 Annealing mR03_#01 with linkerR03 yielded double-stranded primer mR03_#01-L3; annealing mR03_#02 with linkerR03 yielded double-stranded primer mR03_#02-L3; ..., annealing mR03_#96 with linkerR03 yielded double-stranded primer mR03_#96-L3. Reaction conditions: 95°C for 5 minutes; cooling to 4°C at a rate of 0.1°C per second; holding at 4°C.

[0064] Blocking buffer, total volume 1000 μL: 264 μL of R02 blocking oligonucleotide (100 μM, Sangon Biotech), 250 μL of 10× T4 DNA ligase buffer, and 486 μL of sterile water to make up the total volume.

[0065] The stop solution, total volume 764 μL: 264 μL of R02-quenched oligonucleotides (100 μM, Sangon Biotech) and 500 μL of EDTA (0.5 M).

[0066] Example 1. Construction of a high-throughput single-cell DNA methylation and single-cell chromatin structure co-capture library from mouse embryonic stem cells.

[0067] 1. Dissociation of tissues or cell lines.

[0068] 1) Transfer the tissue sample (mouse embryonic stem cells mESC) into a 5 mL tube containing dissociation buffer and mix thoroughly by pipetting.

[0069] 2) Filter the cell suspension using a 40 µm cell filter and collect the filtrate in a 50 mL centrifuge tube.

[0070] 3) Centrifuge the filtered cell suspension at 4°C and 800 g for 5 minutes and discard the supernatant.

[0071] 4) Resuspend the cell pellet in 5 mL of cross-linking buffer.

[0072] 5) Use a cell counter to count the cells.

[0073] 6) Take 10 million to 20 million cells and transfer them to a new 50 mL centrifuge tube to obtain a single-cell suspension for subsequent cross-linking.

[0074] 2. Cell cross-linking to fix chromatin conformation.

[0075] 1) Prepare formaldehyde solution: Add 330 μL of 16% formaldehyde to 10 mL of cross-linking buffer to obtain formaldehyde solution.

[0076] 2) Adjust the volume of the single-cell suspension obtained in step 1(6) to 5 mL using the formaldehyde solution prepared in step 1), with a final formaldehyde concentration of 1%. Fully resuspend the cells and incubate at room temperature for 10 minutes to perform cell cross-linking.

[0077] 3) Add 1.5 mL of 2 M Tris-HCl (pH=7.5) to terminate crosslinking and incubate at room temperature for 5 minutes.

[0078] 4) Centrifuge at 4°C and 1000 g for 5 minutes and discard the supernatant.

[0079] 5) Wash the cell pellet once with 2 mL of PBS containing 0.1% BSA.

[0080] 6) Resuspend the cell pellet in 5 mL of 3 mM DSG solution and incubate at room temperature for 45 minutes by rotation to perform a second cross-linking.

[0081] 7) Add 1.5 mL of 2M Tris-HCl (pH=7.5) and incubate at room temperature for 5 minutes.

[0082] 8) Centrifuge at 4°C and 1000 g for 5 minutes and discard the supernatant.

[0083] 9) Wash the cell pellet once with 2 mL of PBS containing 0.1% BSA.

[0084] 10) Dispense the cells into tubes of 1 million.

[0085] 11) Centrifuge at 4°C and 1000 g for 5 minutes, discard the supernatant, and obtain a cell pellet containing DNA-protein complexes with fixed chromatin conformation.

[0086] 12) Immediately freeze the cell pellet in liquid nitrogen for subsequent MNase digestion.

[0087] 3. Micrococcal nuclease (MNase) digests chromatin.

[0088] 1) Thaw the cell pellet obtained in step 2 (1 million cells per tube) on ice.

[0089] 2) Resuspend each cell pellet in 100 μL LMB1 working solution and incubate on ice for 20 minutes.

[0090] 3) Centrifuge at 4°C and 800 g for 5 minutes and discard the supernatant.

[0091] 4) Resuspend each cell pellet in 100 μL LMB1 working solution (containing digitalis saponins that disrupt cell membranes, turning cell units into nucleus units) to obtain single-cell nucleus suspension 1.

[0092] 5) Take 5 μL of single-cell suspension 2 from each tube as quality control sample 1 (QC1).

[0093] 6) Add 7.5 μL of 200 U / μL MNase working solution stock solution to the remaining single-cell suspension 2 in each tube to make the MNase content in each tube 1500 U (i.e., the amount of micrococcal nuclease used is 1500 U / million cell nuclei), and incubate in a heat mixer at 37°C and 800 rpm for 10 minutes to digest the chromatin in the cell nucleus.

[0094] 7) Add 0.8 μL of 0.5 M EGTA to terminate the reaction, and gently tap the tube wall with your finger to mix.

[0095] 8) Incubate at 58°C for 10 minutes.

[0096] 9) Take 5 μL of the reaction solution from the incubation product of step 8) as quality control sample 2 (QC2).

[0097] 10) Centrifuge the remaining incubation product from step 8) at 4°C and 800 g for 5 minutes and discard the supernatant.

[0098] 11) Dilute 10× NEBuffer 3.1 with sterile water to make 1× working solution.

[0099] 12) Gently resuspend the precipitate in 200 μL of 1× NEBuffer 3.1 (NEB, B7203S) containing 0.1% SDS (Invitrogen, 15553-035) and incubate at 58°C for 10 min, where SDS is used to loosen the chromatin.

[0100] 13) Add 5 μL of freshly prepared 10% Triton X-100 (Sigma, T9823) to quench SDS and mix thoroughly.

[0101] 14) Incubate at 37°C for 10 minutes.

[0102] 15) Centrifuge at 4°C and 800 g for 5 minutes and discard the supernatant.

[0103] 16) Resuspend the cell pellet in 100 μL LMB2.

[0104] 17) Centrifuge at 4°C and 800 g for 5 minutes, discard the supernatant, and obtain a cell pellet containing digested chromatin fragments (DNA fragments-proteins), i.e., genomic fragments (named single-nucleus 1 pellet). The size of the genomic fragments (existing in the form of chromatin fragments) is 100bp-200bp, mainly fragments of about 120bp.

[0105] 4. Chromatin fragment end repair.

[0106] Chromatin fragment end repair involves flattening chromatin fragments to create blunt ends and adding phosphate groups.

[0107] 1) Resuspend each cell pellet in 45 μL of end-repair reaction solution (ECB), gently tap to mix, and incubate in a heat mixer at 37°C and 200 rpm for 15 minutes.

[0108] 2) Then add 5 μL of Klenow fragment (5 U / μL, NEB, M0210L) to each tube, gently tap to mix, and incubate with shaking at 37°C and 200 rpm for 15 minutes in a hot mixer.

[0109] 3) Add 25 μL of end-extension reaction solution (ELB) to each tube, gently tap to mix, and incubate with shaking at 25°C and 200 rpm for 45 minutes in a hot mixer.

[0110] 4) Add 9 μL of 0.5 M EDTA to each tube and gently tap with your finger to mix.

[0111] 5) Incubate at 58°C for 10 minutes.

[0112] 6) Centrifuge at 4°C and 800 g for 5 minutes and discard the supernatant.

[0113] 7) Resuspend the precipitate in 200 μL of pre-cooled MB3 in each tube.

[0114] 8) Centrifuge at 4°C and 800 g for 5 minutes and discard the supernatant.

[0115] 5. Neighbor-to-neighbor connections of chromatin segments.

[0116] 1) Resuspend each precipitate in 250 μL of ligation reaction solution, gently tap to mix, and incubate at room temperature for 4 hours to obtain single-cell nucleus suspension 2 (obtained after ligating chromatin fragments to adjacent ends).

[0117] 2) Take 5 μL of reaction solution from the single-cell nucleus suspension 2 in step 1) as quality control sample 3 (QC3).

[0118] 3) Count the nuclei of the remaining single-nucleus suspension 2 from step 1).

[0119] 4) Divide the single-cell nucleus suspension into 2 portions, each containing approximately 200,000 cells, for a total of 6 portions, to be used for the first round of barcode linking.

[0120] 6. Combined tags are used to link chromatin fragments from different single-cell sources.

[0121] Using a ligation-based combinatorial barcoding strategy, the chromatin fragments in the single-nucleus suspension 2 obtained in step 5 were ligated through three rounds of splitting and pooling. Chromatin fragments from different single-nucleus sources were tagged with different barcode combinations, each combination containing three tandem barcodes (barcode3-barcode2-barcode1): barcode1 (5'-pAGGCCAGAGCATTCGGTTTAGNNNNNNNNACAG-3', sequence 207, where nucleotides 22-29 are used to distinguish different barcodes). The tag sequence for barcode 1; barcode 2 (5'-pGTGCGAACTCAGACCNNNNNNNNTCCACGTGCTTGAG-3', sequence 208, where nucleotides 16-23 are tag sequences distinguishing different barcode 2s) and barcode 3 (5'-GGAGATGTGTATAAGAGAAGNNNNNNNNNNNTGGCCGATGTTTCG-3', sequence 209, where nucleotides 21-28 of sequence 209 are tag sequences distinguishing different barcode 3s, and nucleotides 29-31 are random nucleotide sequences designed to prevent mismatches and improve sequencing efficiency). N is A, T, C, or G.

[0122] 6.1 First round of barcode connection.

[0123] 1) Centrifuge the 6 aliquots of cell nucleus suspension obtained in step 5 at 4°C and 800 g for 5 minutes and discard the supernatant.

[0124] 2) Resuspend one portion of the cell nucleus pellet in 155 μL of the first round barcode ligation reaction solution (BLB#1) to obtain the first round of cell nucleus suspension. Add 35 μL of one of the six corresponding barcode #1 adapters and 10 μL of T4 DNA ligase.

[0125] 3) Gently tap the mixture with your finger to mix, incubate overnight at 16°C, and add the first round of barcode label (barcode1) to obtain a single-cell nucleus suspension 2-1 containing the first round of barcode linking products, that is, a cell nucleus suspension containing chromatin fragments with added barcode1 (barcode1-chromatin fragment).

[0126] 6.2. Composite Index and Sorting.

[0127] 1) Rinse two 15 mL centrifuge tubes with PBS containing 0.1% BSA.

[0128] 2) Combine all single-cell nucleus suspensions 2-1 samples, centrifuge at 4°C and 800 g for 5 minutes, discard the supernatant, and obtain the first round of cell nucleus mixture precipitation.

[0129] 3) Resuspend the first round of cell nucleus mixture precipitate with an appropriate amount of BLB#2 mixture for the second round of barcode linking, and mix well to obtain the second round of single cell nucleus suspension.

[0130] 4) Dispense the second round of cell nucleus suspension into individual cell nuclei using a multichannel pipette into a barcode plate (R02 plate, each well of which is pre-labeled with one of the second round barcodes, barcode2), 30 μL per well, and seal the plate.

[0131] 5) Incubate in a hot mixer at 37°C and 300 rpm for 45 minutes with shaking.

[0132] 6) Use a multichannel pipette to add 10 μL of sealing solution to each well, mix gently, and seal the plate.

[0133] 7) Incubate the mixture in a heat mixer at 37°C and 300 rpm for 15 minutes to obtain a single-cell nucleus suspension 2-2 containing the second round of barcode ligation products, i.e., a cell nucleus suspension containing chromatin fragments with added barcode2-barcode1 (barcode2-barcode1-chromatin fragment).

[0134] 8) Combine the single-cell nucleus suspensions from all wells into a pre-washed 15 mL centrifuge tube.

[0135] 9) Centrifuge at 4°C and 800 g for 5 minutes, discard the supernatant, and obtain the second round of cell nucleus mixture precipitation.

[0136] 10) Resuspend the second round of nuclear mixture precipitate in an appropriate amount of BLB#3 mixture and mix well.

[0137] 11) Dispense individual cell nuclei from the cell nucleus suspension into a barcode plate (R03 plate, each well of which is pre-labeled with one of the third round barcodes, barcode3) using a multichannel pipette, 30 μL per well, and seal the plate.

[0138] 12) Incubate in a hot mixer at 37°C and 300 rpm for 45 minutes with shaking.

[0139] 13) Using a multichannel pipette, add 10 μL of stop solution to each well, mix gently, and add the third round barcode (barcode3) label to obtain single-cell nucleus suspension 2-3 containing the third round barcode ligation product, that is, a cell nucleus suspension containing chromatin fragments with added barcode3-barcode2-barcode1 (barcode3-barcode2-barcode1-chromatin fragment).

[0140] 14) Combine the single-cell nucleus suspensions 2-3 from all wells into a pre-washed 15 mL centrifuge tube.

[0141] 15) Centrifuge at 4°C and 800 g for 5 minutes and discard the supernatant.

[0142] 16) Wash the precipitate once with 200 μL of PBS containing 0.1% BSA to obtain single-cell nucleus suspension 3.

[0143] 17) Count the cell nuclei and aliquot them into sub-libraries, each containing approximately 2000 single nuclei. The sub-libraries can be stored at -80°C.

[0144] 7. Decrosslinking and purification of target DNA.

[0145] 1) Add the following reagents to each sub-library to break the covalent cross-links between the protein and the target DNA, thereby releasing the target DNA fragment (barcode2-barcode1-DNA fragment) for subsequent purification: 3 μL SDS (10%), 3 μL proteinase K (20 mg / mL, Roche, 3115828001), 3 μL NaCl (4 M), and bring the total volume to 30 μL with PBS.

[0146] 2) Incubate in a hot mixer at 62°C and 800 rpm for 2.5 hours with shaking.

[0147] 3) Purify the target DNA fragment (barcode2-barcode1-DNA fragment) using 1.0 volume (30 μL) of SPRI (Beckman, B23318) magnetic beads.

[0148] 4) Elute the target DNA fragment with 28 μL of ultrapure water to obtain the target DNA fragment for each sub-library.

[0149] 8. Base conversion of the target DNA fragment.

[0150] In this embodiment, the cytosine conversion method in enzyme methylation sequencing EM-seq is used to construct a methylation sequencing library (base conversion) of the target DNA fragment of the sub-library obtained in step 7 (including cytosine modification of the target DNA fragment and deamination after cytosine modification).

[0151] 1) Elute the target DNA fragments of each sub-library obtained in step 7 into 39 μL of ultrapure water.

[0152] 2) Prepare an EM-seq (NEB, E7125L) TET2 reaction module (using TET2 enzyme and oxidation enhancer to oxidize methylated cytosine 5mC and 5-hydroxymethylcytosine 5hmC in the target DNA fragment to 5caC, protecting the modified cytosine), react for 1 hour to perform cytosine modification treatment, then add stop buffer and react for 30 minutes to obtain the cytosine-modified target DNA fragment.

[0153] 3) Purify the cytosine-modified target DNA using 1.8 times the volume (90 μL) of SPRI magnetic beads.

[0154] 4) Elute the DNA with 16 μL of ultrapure water.

[0155] 5) Add 4 μL of 0.1N NaOH solution and react at 50°C for 10 minutes.

[0156] 6) Prepare the EM-seq APOBEC reaction module (using the deaminase APOBEC3A to deaminate cytosine; this step will not affect 5caC, thus enabling the detection of 5mC and 5hmC), and react for 5 hours to obtain the deaminated target DNA.

[0157] 7) Purify DNA using 1 volume (100 μL) of SPRI magnetic beads.

[0158] 8) Elute the DNA with 20 μL of ultrapure water to obtain the final target DNA fragment after base conversion.

[0159] 9. Three rounds of PCR amplification were performed to obtain the final sequencing library.

[0160] 1) Add the following to the target DNA fragment after base conversion in each sub-library: 2 μL of dNTP mixture (10 mM, Thermo, R0192), 5 μL of 10× NEBuffer 2.1 (NEB, B7202V), and 2 μL of primer P5-10H (10 μM, Sangon Biotech, sequence 210, 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNN-3', where N is A, T, C, or G, and C is 5caC, 5mC, or 5hmC), where nucleotides 1-31 of sequence 210 are complementary to the P5 sequence of the Illumina sequencing platform.

[0161] 2) Perform the following linear PCR amplification program: 98°C, 2 minutes; immediately place on ice for 3 minutes.

[0162] 3) Add 2 μL of Klenow exo-fragment (NEB, M0212L).

[0163] 4) Perform the following PCR program: 4°C, 5 minutes; increase temperature to 37°C at a rate of 1°C every 15 seconds; 37°C, 60 minutes; 4°C, hold.

[0164] 5) Purify DNA using 1.0 volume (50 μL) of SPRI magnetic beads.

[0165] 6) Elute the DNA with 21 μL of ultrapure water.

[0166] 7) Add the following to the eluted DNA: 2 μL of primer pre-P5 (10 μM, Sangon Biotech, sequence 211: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATC-3'), 2 μL of primer pre-N7 (10 μM, Sangon Biotech, sequence 212: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'), and 25 μL of 2× KAPA HiFi HotStart ReadyMix (Roche, KK2601).

[0167] 8) Perform the following PCR preamplification program: 98°C, 3 min, initial denaturation; 98°C, 10 s, denaturation; 60°C, 30 s, annealing; 72°C, 60 s, extension; repeat 4 cycles; 72°C, 60 s, final extension; 4°C, hold.

[0168] 9) DNA was purified using 0.8 times the volume (40 μL) and 0.8 times + 0.2 times the volume (40 + 10 μL) of SPRI magnetic beads, respectively. Different sizes of magnetic beads were used to screen DNA fragments of different lengths: 0.2 times the volume of SPRI magnetic beads was used to purify methylated DNA fragments, and 0.8 times the volume of SPRI magnetic beads was used to purify chromatin interaction-related DNA fragments.

[0169] 10) Elute the DNA with 22 μL of ultrapure water. Take 1 μL for Qubit quantification.

[0170] 11) Take an appropriate amount of the purified DNA from the previous step and add: 2 μL of primer P5 (10 μM, Sangon Biotech, sequence 213: 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3', where the 70th nucleotide in sequence 213 is a phosphate-thiolated T), 2 μL of primer N7 (10 μM, Sangon Biotech, sequence 214, 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'), and 25 μL of 2× KAPA HiFi HotStart ReadyMix. N in the primers represents A, T, C, or G.

[0171] The four P5 series primers (P5_5-P5_8, corresponding to sequences 215-218 in the sequence listing) and the four N7 series primers (N7_20-N7_24, corresponding to sequences 219-222 in the sequence listing) are listed as follows: Figure 9 .

[0172] 12) Perform the following third round PCR program (with sequencing adapters added): 98°C, 3 min, initial denaturation; 98°C, 10 s, denaturation; 60°C, 30 s, annealing; 72°C, 60 s, extension; repeat 6 cycles; 72°C, 60 s, final extension; 4°C, hold.

[0173] 13) Purify DNA using 0.95 times the volume (47.5 μL) of SPRI magnetic beads.

[0174] 14) Elute with 15-30 μL of ultrapure water to obtain the final sequencing libraries of each sub-library.

[0175] 15) Take 1 μL of the sequencing library from each sub-library for Qubit quantification, take 0.5 μL for qPCR to assess library quality, and then perform sequencing.

[0176] 10. qPCR is used to assess the quality of sequencing libraries.

[0177] The sequencing libraries of the three sub-libraries obtained in step 9 were subjected to qPCR detection using the Novizan NQ105 qPCR kit. The blank control was deionized water, and standards 1-6 were derived from the kit.

[0178] Table 1 shows that the Ct values ​​of the three sub-libraries were all low, while the concentrations were all high, indicating that a high concentration of adapter DNA sequences were added, which is beneficial for sequencing.

[0179] Table 1. qPCR data of sub-libraries for the three tests.

[0180]

[0181] 11. Sequencing and splitting to obtain single-cell level DNA methylation and chromatin structure co-capture sequencing data.

[0182] The sequencing library obtained in step 9 for the next-generation sequencing platform was subjected to next-generation sequencing on the Illumina NovaSeq X Plus platform. The quality control results of the sequencing data are shown using test sub-library 1 as an example (Table 2). The results show that test sub-library 1 has a high Q30 value and sequencing depth, as well as a suitable CG content. The base distribution of this library clearly shows the barcode sequence information of this invention at approximately 100 bp positions corresponding to positions 150-250, proving that the sequencing library constructed in this invention captured the expected target DNA sequence for DNA methylation and chromatin structure co-capture analysis.

[0183] Table 2. Quality control results of the test sub-library.

[0184]

[0185] Sequencing data is split into single-cell data based on barcode combination tags to obtain single-cell level DNA methylation and chromatin structure co-capture sequencing data. The sequencing data analysis workflow mainly includes: 11.1 Tag splitting.

[0186] Combination tags (barcode3-barcode2-barcode1) are extracted from sequencing reads in the sequencing data. Barcodes are located using the linker sequences (Linker01 and Linker02), and barcodes with more than one mismatch in the linker region are discarded. The three rounds of barcode sequences are extracted and mapped to a barcode reference library. Figures 6-8The mapped barcodes are then assigned to the read names of read 1 and read 2 in the paired-end sequencing reads for downstream analysis.

[0187] 11.2 Segment comparison.

[0188] Cutadapt and Trim galore were used to remove adapters from the demultiplexed sequencing reads. This invention employed the Taurus-MH and Bismark software packages to perform two rounds of alignment on read 1 and read 2 of the sequencing reads. Specifically, read 1 and read 2 were aligned to the reference genome (GRCm38), and unaligned reads were collected. Then, read 1 was further trimmed from both sides of read 1 and from the 3' end of read 2. Read 1 was further divided into two or three segments. These unaligned reads were then aligned to the reference genome again. All aligned sequencing reads were merged into BAM format.

[0189] 11.3 Extraction of chromatin structure and methylation information.

[0190] The outermost aligned reads in each fragment were selected as chromatin contact points. Cooler was used to convert all chromatin contact points into cool files suitable for visualization analysis. Methylation information was extracted from all aligned reads using Bismark, resulting in bedGraph files containing genomic methylation distribution information. The resulting chromatin contact and methylation files can then be used for downstream biological information mining.

[0191] 12. Comparative analysis of the method of the present invention (this method) with the prior art.

[0192] 12.1 Comparative analysis of cell throughput and capture efficiency.

[0193] Figure 2 This study compares the parameters detected on mouse embryonic stem cell line mESCs using the method of the present invention (the method described in steps 1 to 11 above) with those detected using other single-cell three-dimensional genomics technologies, single-cell methylation technologies, and co-capture technologies for single-cell DNA methylation and single-cell chromatin structure. Figure 2The upper figure shows a comparison of the method of the present invention with existing single-cell methylation detection technologies (M1 represents scWGBS, M2 represents scRRBS, M3 represents snmC-seq, M4 represents sciMET, M5 represents sciMETv2, M6 represents scBS-seq, M7 represents scMspJI-seq, M8 represents Drop-BS, M9 represents sciEM) and two other existing co-capture technologies for single-cell DNA methylation and single-cell chromatin structure (C1 represents scMethyl-HiC, C2 represents sn-m3C-seq) in terms of cell throughput and methylation signal capture efficiency per cell. Figure 2 The lower figure shows a comparison of the method of the present invention with existing single-cell three-dimensional genome detection technologies (H1 represents scHi-C, H2 represents sci-Hi-C, H3 represents snHi-C, H4 represents Dip-C, H5 represents s3-GCC, H6 represents scHi-C v2, H7 represents snHi-C, H8 represents DropletHi-C, H9 represents scMicro-C, H10 represents scNanoHi-C) and two other existing single-cell DNA methylation and single-cell chromatin structure co-capture technologies (C1 represents scMethyl-HiC, C2 represents sn-m3C-seq) in terms of cell throughput and capture efficiency per cell. Figure 2 The results show that, compared with the prior art, the method of the present invention can achieve a significant increase in cell throughput (from thousands of cells per batch to 80,000 cells per batch), while maintaining a high level of capture efficiency per cell, demonstrating the progress of the method over the prior art. This improves the applicability of the present technology in the co-capture of single-cell DNA methylation and single-cell chromatin structure.

[0194] The literature information corresponding to the existing single-cell three-dimensional genome technology, single-cell methylation technology, and co-capture technology of single-cell DNA methylation and single-cell chromatin structure used in this embodiment is as follows: M1: Farlik M, Sheffield NC, Nuzzo A, Datlinger P et al. Single-cellDNA methylome sequencing and bioinformatic inference of epigenomic cell-statedynamics. Cell Rep. 2015 Mar 3; M2:Guo H, Zhu P, Wu X, Li X et al. Single-cell methylome landscapesof mouse embryonic stem cells and early embryos analyzed using reducedrepresentation bisulfite sequencing. Genome Res. 2013 Dec; M3:Luo C, Keown CL, Kurihara L, Zhou J et al. Single-cell methylomesidentify neuronal subtypes and regulatory elements in mammalian cortex.Science. 2017 Aug 11; M4:Mulqueen RM, Pokholok D, Norberg SJ, Torkenczy KA et al. Highlyscalable generation of DNA methylation profiles in single cells. NatBiotechnol. 2018 Jun; M5:Nichols RV, O’Connell BL, Mulqueen RM, Thomas J et al. High-throughput robust single-cell DNA methylation profiling with sciMETv2. NatCommun. 2022 Dec 9; M6:Smallwood SA, Lee HJ, Angermueller C, Krueger F et al. Single-cellgenome-wide bisulfite sequencing for assessing epigenetic heterogeneity. NatMethods. 2014 Aug; M7:Sen M, Mooijman D, Chialastri A, Boisset JC et al. Strand-specificsingle-cell methylomics reveals distinct modes of DNA demethylation dynamicsduring early mammalian development. Nat Commun. 2021 Feb 25; M8:Zhang Q, Ma S, Liu Z, Zhu B et al. Droplet-based bisulfitesequencing for high-throughput profiling of single-cell DNA methylomes. NatCommun. 2023 Aug 3; M9:Chatterton Z, Lamichhane P, Ahmadi Rastegar D, Fitzpatrick L etal. Single-cell DNA methylation sequencing by combinatorial indexing andenzymatic DNA methylation conversion. Cell Biosci. 2023 Jan 4; C1:Li G, Liu Y, Zhang Y, Kubo N et al. Joint profiling of DNAmethylation and chromatin architecture in single cells. Nat Methods. 2019Oct; C2:Lee DS, Luo C, Zhou J, Chandran S et al. Simultaneous profiling of3D genome structure and DNA methylation in single human cells. Nat Methods.2019 Oct; H1:Nagano T, Lubling Y, Stevens TJ, Schoenfelder S et al. Single-cellHi-C reveals cell-to-cell variability in chromosome structure. Nature. 2013Oct 3; H2:Ramani V, Deng X, Qiu R, Gunderson KL et al. Massively multiplexsingle-cell Hi-C. Nat Methods. 2017 Mar; H3:Flyamer IM, Gassler J, Imakaev M, HB et al. Single-nucleusHi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.Nature. 2017 Apr 6; H4:Tan L, Xing D, Chang CH, Li H et al. Three-dimensional genomestructures of single diploid human cells. Science. 2018 Aug 31; H5:Mulqueen RM, Pokholok D, O’Connell BL, Thornton CA et al. High-content single-cell combinatorial indexing. Nat Biotechnol. 2021 Dec; H6:Nagano T, Lubling Y, C, Dudley C et al. Cell-cycle dynamicsof chromosomal organization at single-cell resolution. Nature. 2017 Jul 6; H7: Stevens TJ, Lando D, Basu S, Atkinson LP et al. 3D structures of individual mammalian genomes studied by single-cell Hi-C. Nature. 2017 Apr 6; H8: Chang L, Xie Y, Taylor B, Wang Z et al. Droplet Hi-C enables scalable, single-cell profiling of chromatin architecture in heterogeneous tissues. Nat Biotechnol. Published online 2024 Oct 18; H9: Wu H, Zhang J, Tan L, Xie XS. Single-cell Micro-C profiles 3D genome structures at high resolution and characterizes multi-enhancer hubs. Nat Genet. 2025 Jul 2; H10: Li W, Lu J, Lu P, Gao Y et al. scNanoHi-C: a single-cell long-read concatemer sequencing method to reveal high-order chromatin structures within individual cells. Nat Methods. 2023 Oct.

[0195] 12.2 Parameter comparison in terms of library construction time and application cost.

[0196] Figure 3This paper compares the method of the present invention with two other existing co-capture technologies for single-cell DNA methylation and single-cell chromatin structure (C1 represents scMethyl-HiC, C2 represents sn-m3C-seq) in terms of library preparation time and cost. It is clear that compared to the two existing technologies, the method of the present invention can significantly reduce the cost (one-fifth of the cost based on the same throughput of 10,000 cells) and time (one-third of the time based on the same throughput of 10,000 cells). The method of the present invention enables the co-capture of single-cell DNA methylation and single-cell chromatin structure in a high-throughput, rapid, and low-cost manner, providing a convenient technical tool for single-cell epigenetic status detection based on this co-capture technology.

[0197] Figure 4 This image shows a comparison of chromatin structures captured by sequencing libraries obtained using the method of this invention and signals from common chromatin capture techniques on mouse embryonic stem cells (mESC) chromosome 1. The left image shows the chromatin structure at 500 kb resolution, and the right image shows the chromatin structure at 50 kb resolution. The upper right of both images shows the chromatin structure captured by this method, and the lower left image shows other mass cell capture techniques. Figure 4 The chromatin structures captured by other reference methods (represented in the literature) are clearly visible at different resolutions. The chromatin structure maps captured by the present invention and other numerous cell capture technologies (MicroC, related literature: Hsieh TS, Cattoglio C, Slobodyanyuk E, Hansen AS et al. Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding. Mol Cell. 2020 May 7) for mouse embryonic stem cell mESC samples are similar, demonstrating that the method of the present invention captures the expected chromatin structure.

[0198] Figure 5The results compare the DNA methylation levels captured by the sequencing library obtained using the method of this invention with the methylation modification levels detected by common DNA methylation capture techniques (WGBS, related literature: Ehsan Habibi, et al. Whole-Genome Bisulfite Sequencing of Two Distinct Interconvertible DNA Methylomes of Mouse Embryonic Stem Cells. Cell Stem Cell. 2013) on chromosome 1 of mouse embryonic stem cell mESC samples. The upper figure shows the methylation level changes in the 100-110 Mb region using this method, and the lower figure shows the methylation level changes in the 100-110 Mb region using other numerous DNA methylation capture techniques. It is clear that this invention and other numerous DNA methylation capture techniques have similar methylation level changes, proving that this invention captures the expected DNA methylation signal.

[0199] Example 2. Analysis of brain cell clustering and developmental trajectory in human embryos at 23 weeks of gestation based on the method of the present invention.

[0200] In this embodiment, after comprehensively considering factors such as testing costs and necessity, high-throughput single-cell DNA methylation and single-cell chromatin structure co-capture library construction and sequencing analysis were performed on approximately 10,000 human embryonic brain cells (cerebral cortex cells from aborted human embryos at 23 weeks of gestation, sourced from Beijing Obstetrics and Gynecology Hospital affiliated with Capital Medical University, which has undergone ethical review by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital affiliated with Capital Medical University, and complies with relevant ethical review laws and regulations, with the ethical review decision document number 2023-KY-018-01) to demonstrate the feasibility and applicability of this method in complex tissues.

[0201] 1. Dissociation of tissues or cell lines.

[0202] 1) Embed the tissue sample with OCT and perform cryosection on a cryostat. Take about 3-5 100 µm sections and transfer them to a 5 mL tube containing dissociation buffer. Mix them thoroughly by repeatedly pipetting.

[0203] 2) Filter the cell suspension using a 40 µm cell filter and collect the filtrate in a 50 mL centrifuge tube.

[0204] 3) Centrifuge the filtered cell suspension at 4°C and 800 g for 5 minutes and discard the supernatant.

[0205] 4) Resuspend the cell pellet in 5 mL of cross-linking buffer.

[0206] 5) Use a cell counter to count the cells.

[0207] 6) Take 10 million to 20 million cells and transfer them to a new 50 mL centrifuge tube to obtain a single-cell suspension for subsequent cross-linking.

[0208] The experimental steps in subsequent steps 2-11 are the same as those in steps 2-11 of Example 1.

[0209] After sequencing data analysis of the sequencing library, the results are as follows: Figure 10 and Figure 11 As shown.

[0210] Figure 10 This invention utilizes the method of the present invention to perform co-capture mapping of single-cell DNA methylation and single-cell chromatin structure in cerebral cortex cells of human embryos at 23 weeks of gestation. Based on the co-capture results, the heterogeneity of gene methylation level distribution in different cells was analyzed and compared. Different types of cerebral cortex cells were clustered and annotated, successfully identifying various cell types including neural progenitor cells, oligodendrocyte precursor cells, microglia, various excitatory neurons, various inhibitory neurons, and perivascular cells. These results demonstrate the co-capture capability of the method of the present invention for DNA methylation and chromatin structure in complex tissues. The single-cell epigenetic detection results obtained based on the method of the present invention can be used for heterogeneity analysis of complex tissue cells such as nerve cells, enabling cell clustering and annotation.

[0211] Figure 11 To utilize the method of this invention for single-cell DNA methylation and single-cell chromatin structure capture of human embryonic cerebral cortex cells at 23 weeks of gestation, developmental trajectory maps of excitatory and inhibitory neurons in the cerebral cortex cells were obtained. These trajectories, from early cell types such as neural progenitor cells to mature excitatory and inhibitory neurons, demonstrate the ability of the method of this invention to identify developmental maturity by extracting epigenetic signals from complex tissues. Figure 11 The left-middle image shows the developmental trajectory of excitatory neurons. This trajectory begins with early cell types, such as neural progenitor cells, and proceeds through immature excitatory neurons, superficial and deep L2 / 3 excitatory neurons, L4 excitatory neurons, L5 excitatory neurons, and finally the most mature L6 excitatory neurons. Figure 11 The right-middle image shows the developmental trajectory of inhibitory neurons. This trajectory begins with early cell types, such as neural progenitor cells, progresses through relatively mature CGE inhibitory neurons, and finally reaches the most mature MGE inhibitory neurons. The neural developmental trajectory illustrates the gradual increase in maturity and maturation of both excitatory and inhibitory neurons.

[0212] The neural development trajectory constructed using the method of this invention can establish a reference assessment system for the degree of epigenetic neural differentiation and developmental maturity. By extracting the epigenetic characteristics of a specified sample and comparing them with the developmental trajectory of a normal sample, the system outputs a developmental deviation index and a maturity score to score the neural development status, thereby achieving an objective evaluation of the neural development status of the sample and screening for abnormally deviating samples.

[0213] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing a sequencing library for detecting DNA methylation and chromatin structure in single cells, comprising cross-linking dissociated single cells or single cell nuclei, genome fragmentation, and chromatin fragment end repair to obtain chromatin fragment end-repaired single cell nuclei or single cells 1, characterized in that: The genome fragmentation is performed using micrococcal nuclease, and the method further includes: 1) Use DNA ligase to perform chromatin fragment ligation on single cell nuclei or single cell 1 to obtain single cell nuclei or single cell 2; 2) Each single cell nucleus or single cell 2 is labeled with different barcode combinations to distinguish the DNA from different single cell nuclei or single cells, resulting in single cell nuclei or single cells 3; 3) Decrosslink all single-cell nuclei or single-cell cells and collect DNA fragments; 4) Construct a methylation sequencing library from the DNA fragment to obtain the base-converted fragment, and obtain a sequencing library for detecting DNA methylation and chromatin structure in single cells.

2. The method according to claim 1, characterized in that: In the genome fragmentation, the amount of micrococcal nuclease used is 1500 U / million cell nuclei or cells.

3. The method according to claim 1 or 2, characterized in that: The methylation sequencing library was constructed using an enzymatic methylation sequencing library construction method.

4. The method according to claim 1 or 2, characterized in that: The different barcode combination labels are implemented using a connection-based combination labeling method.

5. The method according to claim 1 or 2, characterized in that: The method further includes a step of PCR amplification of the base-converted fragment using primers with sequencing platform adapter sequences.

6. The method according to claim 1 or 2, characterized in that: Each of the different barcode combination tags is composed of barcode1, barcode2, and barcode3; barcode1 is a double-stranded DNA obtained by annealing any of the following single-stranded nucleotide pairs: single-stranded nucleotide pairs composed of sequences 1 and 7, sequences 2 and 8, sequences 3 and 9, sequences 4 and 10, sequences 5 and 11, and sequences 6 and 12; barcode2 is any of the 96 double-stranded DNAs obtained by annealing any one of the 96 single-stranded DNAs from sequences 13 to 108 with the single-stranded DNA shown in sequence 205; barcode3 is any of the 96 double-stranded DNAs obtained by annealing any one of the 96 single-stranded DNAs from sequences 109 to 204 with the single-stranded DNA shown in sequence 206.

7. A composition for constructing sequencing libraries for detecting single-cell DNA methylation and chromatin structure, characterized in that: The composition comprises the different barcode combination tags as described in claim 6 and micrococcal nuclease.

8. The composition according to claim 7, characterized in that: The composition further includes at least one of the following substances: DNA ligase, formaldehyde, SDS, proteinase K, TET2 enzyme, and deaminase.

9. The application of the method in preparing products for detecting cell developmental trajectories, preparing products for screening abnormally developing cells, preparing cell clustering products, or preparing products for tracking cell differentiation pathways, characterized in that: The method is the method described in any one of claims 1-6.

10. The application of the composition in constructing sequencing libraries for detecting single-cell DNA methylation and chromatin structure, characterized in that: The composition is the composition according to claim 7 or 8.

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