A method for constructing a single-cell whole-genome methylation and transcriptome dual-omics library

Abstract

This invention discloses a method for constructing a dual-omics library based on single-cell whole-genome methylation and transcriptomics, belonging to the field of sequencing technology. The method includes: lysing single cells in a sorting tube pre-filled with lysis buffer; applying a nucleophilic coating to the inner wall of the sorting tube to allow cell nuclei to adsorb and obtain a supernatant containing mRNA; reverse transcribing the supernatant to obtain cDNA and constructing a transcriptome library; lysing the cell nucleus to release DNA and converting it to bisulfite; ligating the converted DNA with a molecularly tagged adapter to obtain methylation intermediates; treating with exonucleases; merging intermediates with different molecular tags; purifying with magnetic beads, single-stranding, ligating the other end with a sequencing adapter, and amplifying and purifying to obtain a methylated library. This invention achieves simultaneous separation of RNA and DNA in the same tube for library construction, reducing separation and transfer steps, minimizing RNA degradation and nucleic acid loss, and improving the throughput of methylation library construction and the consistency of dual-omics data.

Description

Technical Field

[0001] This invention relates to the field of sequencing methods, and in particular to a method for constructing a dual-omics library based on single-cell whole-genome methylation and transcriptomics. Background Technology

[0002] Combined analysis of genome-wide DNA methylation and transcriptome at the single-cell level helps to correlate epigenetic states and gene expression characteristics at the same cellular scale, providing crucial support for the analysis of cellular heterogeneity and the study of developmental and disease mechanisms. Existing dual-omics library construction protocols typically require obtaining DNA and RNA separately at the single-cell level and completing the library construction processes for methylation libraries and transcriptome libraries separately.

[0003] However, existing technologies generally suffer from lengthy processes and numerous steps in the separation of DNA and RNA from single cells. Especially under conditions of ultra-small sample volumes, multiple transfers and washing operations can easily introduce the risk of RNA degradation, leading to a decrease in the stability and reproducibility of transcriptome-side library construction. At the same time, some schemes rely on magnetic beads to adsorb cell nuclei or to achieve DNA / RNA separation after washing magnetic beads and ligating primers. This makes it difficult to avoid non-specific adsorption of nucleic acids by magnetic beads, resulting in problems such as nucleic acid loss and decreased library complexity.

[0004] On the other hand, single-cell whole-genome methylation library construction usually still requires steps such as adapter ligation, amplification and purification to be completed for each sample after bisulfite conversion. The single-sample experiment cycle is long and the overall efficiency is low, which makes it difficult to meet the high-throughput processing requirements of batch samples, resulting in high time costs and experimental resource consumption. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a method for constructing dual-omics libraries based on single-cell whole-genome methylation and transcriptomics. By coating the inner wall of a sorting tube containing pre-prepared lysis buffer with a 0.01% poly-L-lysine nucleophilic coating, the cell nuclei directly adsorb onto the tube wall after single-cell lysis, separating from the mRNA-containing supernatant. This achieves rapid physical separation of DNA and RNA within the same reaction vessel, reducing steps such as magnetic bead adsorption, centrifugation, washing, and multiple transfers, thus lowering the risk of ultra-small RNA degradation and nucleic acid loss, and improving the stability and reproducibility of transcriptome library construction. Furthermore, a molecular tag is introduced into the methylation side after bisulfite conversion. Intermediate products from multiple single-cell methylation libraries with different molecular tags are merged and uniformly amplified and purified, enabling batch construction of single-cell methylation libraries. This significantly reduces the time and reagent consumption associated with repetitive single-sample operations, improving overall library throughput and experimental efficiency. Therefore, whole-genome methylation libraries and transcriptome libraries can be efficiently obtained at the single-cell scale, providing a reliable data foundation for dual-omics association analysis.

[0006] To achieve the above objectives, this invention provides a method for constructing a dual-omics library of single-cell whole-genome methylation and transcriptomics, comprising:

[0007] To prepare a single-cell sorting 8-well tube or 96-well plate with a poly-L-lysine nucleophilic coating, add 10 μL of 0.01% poly-L-lysine sterile solution to the sorting tube and treat it at 37°C for 5 minutes in a nuclease-free, sterile oven to allow the coating to cure rapidly.

[0008] Single cells are added to a sorting tube containing pre-prepared lysis buffer for lysis. The inner wall of the sorting tube is coated with a poly-L-lysine nucleophilic coating for adsorbing cell nuclei, so that cell nuclei are adsorbed onto the inner wall of the sorting tube and a supernatant containing mRNA is formed.

[0009] The supernatant was subjected to reverse transcription to obtain cDNA, and a single-cell transcriptome library was constructed based on the cDNA.

[0010] The cell nuclei adsorbed on the inner wall of the sorting tube are lysed to release DNA, and bisulfite conversion is performed based on the DNA;

[0011] Linking bisulfite-converted DNA with adapters containing molecularly tagged sequences to obtain molecularly tagged methylated library intermediates;

[0012] Single-cell whole-genome methylation libraries are constructed by merging intermediate products of at least two single-cell methylation libraries with different molecular tag sequences and then amplifying and purifying them.

[0013] In the above technical solution, preferably, the sorting tube is made of a low-adsorption material, and its inner wall is formed with the nucleophilic coating for adsorbing cell nuclei, and the single cell is added into the sorting tube by flow cytometry.

[0014] In the above technical solution, preferably, the specific process of adding a single cell to the sorting tube for lysis includes:

[0015] The sorting tube is shaken and mixed to achieve cell lysis and promote cell nucleus adsorption onto the inner wall of the sorting tube.

[0016] In the above technical solution, preferably, the specific process of taking the supernatant for reverse transcription reaction includes: taking the supernatant directly into the reverse transcription reaction system while the cell nucleus remains adsorbed to the inner wall of the sorting tube.

[0017] In the above technical solution, preferably, the specific process of nuclear lysis of the cell nuclei adsorbed on the inner wall of the sorting tube includes: adding nuclear lysis solution and proteinase K to the sorting tube to react and release the DNA.

[0018] In the above technical solution, preferably, after introducing the adapter of the molecular tag sequence into the DNA converted by bisulfite, and before merging at least two single-cell methylated library intermediates, the methylated library intermediates are further subjected to exonuclease treatment to remove unconnected adapters.

[0019] In the above technical solution, preferably, the specific process of merging the single-cell methylated library intermediates includes: merging 8 single-cell methylated library intermediates with different introduced molecular tag sequences, and then purifying them with magnetic beads after merging.

[0020] In the above technical solution, preferably, the specific process of constructing the single-cell whole-genome methylation library includes:

[0021] The combined products purified by magnetic beads were subjected to high-temperature denaturation and then rapidly placed on ice to obtain denatured single-stranded DNA products.

[0022] Connect the other end of the sequencing adapter to the single-stranded DNA product.

[0023] The products after ligation of sequencing adapters were subjected to library amplification and product purification.

[0024] In the above technical solution, preferably, constructing a single-cell transcriptome library based on the cDNA includes:

[0025] The cDNA was subjected to quality testing, fragmentation, adapter ligation, library amplification, and purification.

[0026] In the above technical solution, preferably, the dual-omics library construction method further includes:

[0027] The concentrations of the single-cell whole-genome methylation library and the single-cell transcriptome library were determined using a Qubit fluorescence quantitative quantitation system.

[0028] The distribution of nucleic acid fragments in the library was detected and quality controlled using a 4200 bioanalyzer.

[0029] The single-cell whole-genome methylation library and the single-cell transcriptome library were subjected to PE150 sequencing and the sequencing results were analyzed.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) By setting a nucleophilic coating on the inner wall of the sorting tube with pre-prepared lysis buffer, the cell nucleus after single cell lysis is directly adsorbed onto the tube wall and separated from the supernatant containing mRNA, thus completing nucleic acid phase separation in the same container, reducing centrifugation, magnetic bead adsorption and multiple transfer operations, and reducing the risk of RNA degradation and DNA / RNA cross-contamination under ultra-micro sample conditions.

[0032] (2) By directly taking the supernatant while the cell nucleus is in an adsorbed state for reverse transcription and constructing a transcriptome library, the RNA exposure time and processing path are shortened, sample loss and batch-to-batch differences are reduced, and the stability, library complexity and data reproducibility of transcriptome-side library construction are improved.

[0033] (3) By performing nuclear lysis of the adsorbed cell nucleus to release DNA and converting it with bisulfite, the methylation information at the single-cell level can be effectively obtained; after conversion, a connector containing a molecular tag sequence is connected to make the single-cell methylation product have a traceable sample identification, providing a basis for subsequent merging processing.

[0034] (4) By merging the intermediate products of at least two single-cell methylated libraries with different molecular tag sequences and then amplifying and purifying them in a unified manner, the steps of repeated library construction for each sample and the time cost are reduced, the consumption of reagents and the accumulation of operational errors are reduced, and the batch processing and throughput of methylated libraries are improved.

[0035] (5) By performing quality control on the concentration and fragment distribution of methylated libraries and transcriptome libraries and conducting sequencing and analysis, the consistency of library quality and the reliability of downstream data are ensured, thereby supporting the joint analysis and correlation study of methylation and expression at the single-cell scale. Attached Figure Description

[0036] Figure 1 This is a flowchart illustrating a method for constructing a dual-omics library of single-cell whole-genome methylation and transcriptomics according to an embodiment of the present invention.

[0037] Figure 2 This is a quality control image of full-length cDNA in a single-cell transcriptome experiment disclosed in one embodiment of the present invention using a 4200 analyzer.

[0038] Figure 3 This is a quality control image of a 4200 analyzer for a Chinese database used in a single-cell transcriptome experiment, as disclosed in one embodiment of the present invention.

[0039] Figure 4 This is a quality control image of a 4200 analyzer for a single-cell whole-genome methylation experimental Chinese library disclosed in one embodiment of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The present invention will now be described in further detail with reference to the accompanying drawings:

[0042] like Figure 1 As shown, a method for constructing a dual-omics library of single-cell whole-genome methylation and transcriptomics according to the present invention includes:

[0043] To prepare a single-cell sorting 8-well tube or 96-well plate with a poly-L-lysine nucleophilic coating, add 10 μL of 0.01% poly-L-lysine sterile solution to the sorting tube and treat it at 37°C for 5 minutes in a nuclease-free, sterile oven to allow the coating to cure rapidly.

[0044] Single cells are added to a sorting tube containing pre-prepared lysis buffer for lysis. The inner wall of the sorting tube is coated with a poly-L-lysine nucleophilic coating for adsorbing cell nuclei. The nucleophilic coating adsorbs cell nuclei that remain structurally intact after lysis, causing the cell nuclei to adhere to the inner wall of the sorting tube and separate from the liquid phase inside the tube, forming a supernatant containing mRNA.

[0045] In this separated state, the supernatant is kept as a liquid system containing mRNA. The supernatant is used for reverse transcription to obtain cDNA, and a single-cell transcriptome library is constructed based on the cDNA, shortening the exposure and processing path of ultra-micro RNA in the open environment.

[0046] After the supernatant is removed, the cell nuclei adsorbed on the inner wall of the sorting tube remain in the original sorting tube for nuclear lysis. The DNA released after nuclear lysis directly enters the bisulfite conversion process, which converts the DNA methylation information into sequence differences that can be used to build a library.

[0047] Link adapters containing molecularly tagged sequences to bisulfite-converted DNA to obtain molecularly tagged methylated library intermediates, and write single-cell origin information into the methylated library intermediates.

[0048] By merging intermediate products from at least two single-cell methylation libraries with different molecular tag sequences and performing unified amplification and purification operations, a single-cell whole-genome methylation library can be constructed, enabling parallel library construction of samples from the same batch.

[0049] In this embodiment, the technical approach of adsorbing cell nuclei with nucleophilic coating, direct reverse transcription of supernatant, in situ transformation and tagging of nuclear DNA, and multi-sample merging amplification and purification reduces the cumbersome operation steps in the traditional DNA / RNA separation process, lowers the risk of ultra-micro RNA degradation and nucleic acid loss, and improves the efficiency of single-cell whole genome methylation library construction through merging treatment, while shortening the experimental cycle.

[0050] In the above embodiments, preferably, the sorting tube is made of a low-adsorption material, which reduces the non-specific adsorption of nucleic acids and proteins by the tube material; at the same time, a nucleophilic coating is formed on its inner wall for adsorbing cell nuclei, so that the adsorption occurs mainly at the cell nucleus, forming an interface characteristic of low non-specific adsorption and directional adsorption of cell nuclei. Single cells are immediately lysed after being flow-sorted into the sorting tube containing pre-prepared lysis buffer, reducing the time delay and sample loss caused by transferring the cells to the lysis container after sorting. Combined with flow cytometry, this enables precise single-cell loading and rapid entry into the lysed / separated state, improving batch-to-batch consistency and reproducibility of dual-omics library construction.

[0051] In the above embodiments, preferably, the specific process of adding single cells into the sorting tube for lysis includes:

[0052] After adding a single cell to the sorting tube, the tube is shaken to mix it, which allows the lysis buffer to come into full contact with the cell and accelerates cell membrane rupture, shortening the lysis time window. At the same time, the fluid shear and convection caused by shaking increases the frequency of contact between the cell nucleus and the nucleophilic coating on the tube wall, allowing the cell nucleus to be more quickly and stably adsorbed onto the inner wall, and establishing a separated state of cell nucleus adsorption and supernatant containing mRNA as soon as possible.

[0053] In this embodiment, oscillation and mixing simultaneously enhance lysis efficiency and the rate of nuclear adsorption establishment, reduce waiting time, lower the risk of mRNA degradation due to prolonged residence in the lysis system, and improve the stability of subsequent reverse transcription input.

[0054] In the above embodiments, preferably, the specific process of taking the supernatant for reverse transcription includes: with the cell nuclei still adsorbed to the inner wall of the sorting tube, the supernatant is directly taken from the sorting tube and introduced into the reverse transcription reaction system. Since the cell nuclei are fixed to the tube wall, the entrainment of DNA components during the supernatant sampling process is reduced, and the mRNA background in the reverse transcription system is more concentrated, which is conducive to increasing the effective template ratio of the reverse transcription reaction, reducing the steps of magnetic bead adsorption, washing separation and multiple transfers, shortening the operation link and reducing the risk of ultra-micro RNA degradation and cross-contamination, thereby improving the stability and data reproducibility of transcriptome library construction.

[0055] In the above embodiments, preferably, the specific process of nuclear lysis of cell nuclei adsorbed on the inner wall of the sorting tube includes: adding nuclear lysis solution to the cell nuclei adsorbed on the inner wall of the sorting tube and adding proteinase K to react.

[0056] Specifically, the nuclear lysis buffer disrupts the nuclear membrane structure and releases nuclear DNA, while proteinase K degrades nuclear proteins, reducing conversion and library construction inhibition caused by DNA-protein complexation. This allows the released DNA to enter the subsequent bisulfite conversion step in a more suitable form. The synergistic effect of the nuclear lysis buffer and proteinase K improves DNA release efficiency and reduces protein interference, enhancing the stability of bisulfite conversion and subsequent adapter ligation, and improving the complexity and effective read ratio of single-cell methylated libraries.

[0057] In the above embodiments, preferably, after introducing the adapter of the molecular tag sequence into the DNA converted by bisulfite, and before merging at least two single-cell methylated library intermediates, the methylated library intermediates are further subjected to exonuclease treatment to remove unligated adapters.

[0058] Specifically, this exonuclease treatment removes unligated adapters, significantly reducing the number of free adapters in the subsequent merging system and lowering the probability of adapter dimer and non-specific product formation during amplification. Performing exonuclease cleanup before merging improves the purity of the merged amplification system, reduces the proportion of non-specific amplification, and enhances the proportion of effective insert fragments and sequencing output quality after library preparation.

[0059] In the above embodiments, preferably, the specific process of merging the intermediate products of the single-cell methylated library includes: selecting 8 single-cell methylated library intermediate products with different introduced molecular tag sequences for merging, and then performing magnetic bead purification after merging.

[0060] Specifically, the merging operation utilizes molecular tags to achieve sample traceability, while centralizing the purification steps that were originally performed sample by sample, significantly improving the efficiency of single-cell whole-genome methylation experiments and shortening the average experimental cycle; magnetic bead purification is used to remove salt ions, small molecule residues and short fragment impurities, so that the merged products meet the subsequent purity requirements, reduce intra-batch variability, and improve the success rate of subsequent library construction.

[0061] In the above embodiments, preferably, the specific process of constructing a single-cell whole-genome methylation library includes:

[0062] The combined products purified by magnetic beads were subjected to high-temperature denaturation and then rapidly placed on ice to obtain denatured single-stranded DNA products.

[0063] By ligating the other end of the sequencing adapter onto the single-stranded DNA product, the reaction interface for ligating the other end of the sequencing adapter onto the single-stranded template becomes more consistent, enabling the methylated library to form a sequenceable double-ended structure and reducing the impact of the double-stranded structure on the accessibility of the adapter ligation site.

[0064] After completing the ligation of the other end of the sequencing adapter, the product is amplified and purified to increase the usable amount of the library and reduce the background impurities, so that the library concentration and fragment purity meet the requirements for sequencing, thereby improving the success rate of sequencing library construction and the stability of read quality.

[0065] In the above embodiments, preferably, constructing a single-cell transcriptome library based on cDNA includes:

[0066] After obtaining cDNA through reverse transcription, the cDNA is subjected to quality testing to confirm that the cDNA yield and fragment distribution meet the requirements for subsequent library construction.

[0067] The cDNA is then fragmented to ensure the insert length fits within the sequencing platform's adaptation range. The fragmented cDNA is then ligated with adapters to form an amplifiable structure. Library amplification and purification are then performed to remove small molecules and short fragment byproducts.

[0068] Specifically, by implementing closed-loop control of cDNA quality control, fragmentation, adapter ligation, and amplification purification, the risk of failure caused by direct library construction with low-quality cDNA is reduced, the complexity and fragment distribution consistency of single-cell transcriptome libraries are improved, and the data comparability when analyzed in conjunction with methylated libraries is enhanced.

[0069] In the above embodiments, preferably, the dual-omics library construction method further includes:

[0070] The concentrations of single-cell whole-genome methylation libraries and single-cell transcriptome libraries were determined using a quantitative fluorescence detection kit (such as the Qubit dsDNA HS Assay Kit) to provide a quantitative basis for the library loading capacity.

[0071] The distribution of nucleic acid fragments in the library was detected and quality controlled using a 4200 bioanalyzer to confirm that the position of the main peak and the background of short fragments met the sequencing requirements.

[0072] PE150 sequencing and sequencing results analysis were performed on single-cell whole-genome methylation libraries and single-cell transcriptome libraries. In the sequencing results analysis, the methylation library was used to differentiate samples based on molecular tags and methylation information was analyzed, while the transcriptome library was used to analyze expression levels, resulting in dual-omics data output at the single-cell scale.

[0073] In this implementation, the closed-loop link of quantification, fragment distribution and sequencing analysis ensures the consistency of the quality of the two types of libraries on the sequencing machine, and enables methylation information and expression information to be used together for downstream association analysis at the single-cell level, thereby improving the availability and stability of dual-omics data.

[0074] The method for constructing a dual-omics library of single-cell whole-genome methylation and transcriptome disclosed in the above embodiments is illustrated in the following examples, which describe the specific process and effects.

[0075] Example 1:

[0076] Single cells were added to sorting tubes containing pre-prepared lysis buffer for lysis. The lysis buffer was prepared as follows:

[0077]

[0078] The inner wall of the sorting tube is coated with a nucleophilic coating for adsorbing cell nuclei, so that the cell nuclei are adsorbed onto the inner wall of the sorting tube and form a supernatant containing mRNA.

[0079] The supernatant was used for reverse transcription to obtain cDNA.

[0080] The reverse transcription reaction system is prepared as follows:

[0081]

[0082] Gently blow and mix thoroughly, avoiding air bubbles. Collect the liquid at room temperature (700g) to the bottom of the tube.

[0083] The program runs as follows:

[0084]

[0085] The following reagents are added during the pre-amplification process:

[0086]

[0087] After adding 30 μL of PCR MIX to the above reaction solution, the total system was 50 μL. After vortexing and mixing, the liquid was collected at room temperature and 700g to the bottom of the tube.

[0088] The program runs as follows:

[0089]

[0090] Store PCR products at -20°C or -80°C.

[0091] The cDNA magnetic bead purification process includes:

[0092] Add 1×Ampure XP beads (50ul) to each sample, mix by pipetting 10 times, and incubate at room temperature for 8min.

[0093] Place on a magnetic rack for 5 minutes until the liquid becomes clear, discard the supernatant, and wash twice with 200 μL of 80% ethanol;

[0094] After drying, add 17.5uLEB (or NFH2O) to dissolve, mix by pipetting 10 times, incubate for 2 minutes, place on a magnetic rack for 2 minutes, and aspirate 15ul of elution buffer into a new 0.2ml PCR tube.

[0095] like Figure 2 As shown, a qualified cDNA fragment is larger than 1000bp and has no small fragment spikes.

[0096] cDNA fragmentation includes:

[0097] The following reaction system was prepared on ice:

[0098]

[0099] The program runs as follows:

[0100]

[0101] Tn5 enzyme inactivation: Add 5 μL of NT buffer to the above reaction system, mix well by pipetting, and incubate at room temperature for 5 min.

[0102] The following reagents were used for library amplification:

[0103]

[0104] The program runs as follows:

[0105]

[0106] The purification process of the library product includes:

[0107] Purify 0.6×Ampure XP beads (30ul), dissolve in 17.5 uLEB (or NFH2O), and aspirate 15ul of eluent into a new 1.5ml low-adsorption EP tube.

[0108] The transcriptome library quality control process includes:

[0109] like Figure 3 As shown, Qubit was used to measure concentration, and 4200 was used to detect fragment distribution. The average fragment peak of the qualified library was 300-400bp.

[0110] Lysing cell nuclei to construct whole-genome methylation libraries;

[0111] The cell nuclei adsorbed on the inner wall of the sorting tube were lysed to release DNA. The lysis system is as follows:

[0112]

[0113] The program runs as follows:

[0114]

[0115] Add 0.5 µL of 20 mg / mL proteinase K and incubate in a PCR instrument. The program is as follows:

[0116]

[0117] And based on DNA, bisulfite conversion is performed. The conversion system is as follows:

[0118]

[0119] The reaction procedure is as follows:

[0120]

[0121] Linking bisulfite-converted DNA with adapters containing molecularly tagged sequences yields methylated library intermediates with molecular tags:

[0122]

[0123] Template pre-amplification includes:

[0124]

[0125] The program runs as follows:

[0126]

[0127] After completion, add 3 μL of exonuclease I and combine the products for magnetic bead purification.

[0128] Single-cell whole-genome methylation libraries are constructed by merging intermediate products of at least two single-cell methylation libraries with different molecular tag sequences and then amplifying and purifying them.

[0129] 3' end connector connection

[0130]

[0131] The program runs as follows:

[0132]

[0133] Library augmentation specifically includes:

[0134]

[0135] The program runs as follows:

[0136]

[0137] After library amplification, purification was performed using 0.8X magnetic beads, concentration was measured using Qubit, and fragment distribution was detected using 4200. Figure 4 As shown, the average fragment peaks of qualified libraries are in the range of 400-1000bp.

[0138] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a dual-omic library of single-cell whole-genome methylation and transcriptome, characterized in that, include: To prepare a single-cell sorting 8-well tube or 96-well plate with a poly-L-lysine nucleophilic coating, add 10 μL of 0.01% poly-L-lysine sterile solution to the sorting tube and treat it at 37°C for 5 minutes in a nuclease-free, sterile oven to allow the coating to cure rapidly. Single cells are added to a sorting tube containing pre-prepared lysis buffer for lysis. The inner wall of the sorting tube is coated with a poly-L-lysine nucleophilic coating for adsorbing cell nuclei, so that cell nuclei are adsorbed onto the inner wall of the sorting tube and a supernatant containing mRNA is formed. The supernatant was subjected to reverse transcription to obtain cDNA, and a single-cell transcriptome library was constructed based on the cDNA. The cell nuclei adsorbed on the inner wall of the sorting tube are lysed to release DNA, and bisulfite conversion is performed based on the DNA; Linking bisulfite-converted DNA with adapters containing molecularly tagged sequences to obtain molecularly tagged methylated library intermediates; Single-cell whole-genome methylation libraries are constructed by merging intermediate products of at least two single-cell methylation libraries with different molecular tag sequences and then amplifying and purifying them.

2. The dual-omics library construction method of claim 1, wherein, The inner wall of the sorting tube is formed with a nucleophilic coating for adsorbing cell nuclei, and the single cell is added into the sorting tube by flow cytometry.

3. The dual-omics library construction method of claim 1, wherein, The specific process of adding a single cell into the sorting tube for lysis includes: The sorting tube is shaken and mixed to achieve cell lysis and promote cell nucleus adsorption onto the inner wall of the sorting tube.

4. The dual-omics library construction method of claim 1, wherein, The specific process of taking the supernatant for reverse transcription includes: taking the supernatant directly into the reverse transcription reaction system while the cell nuclei remain adsorbed to the inner wall of the sorting tube.

5. The method for constructing a dual-omics library according to claim 1, characterized in that, The specific process of lysing cell nuclei adsorbed on the inner wall of the sorting tube includes: adding nuclear lysis buffer and proteinase K to the sorting tube to react and release the DNA.

6. The dual-omics library construction method according to claim 1, characterized in that, After introducing a molecular tag sequence adapter into the bisulfite-converted DNA, and before merging at least two single-cell methylated library intermediates, the method further includes exonuclease treatment of the methylated library intermediates to remove unligated adapters.

7. The dual-omics library construction method according to claim 6, characterized in that, The specific process of merging the intermediate products of the single-cell methylated library includes: merging eight single-cell methylated library intermediate products with different introduced molecular tag sequences, and then purifying them with magnetic beads after merging.

8. The method for constructing a dual-omics library according to claim 7, characterized in that, The specific process of constructing the single-cell whole-genome methylation library includes: The combined products purified by magnetic beads were subjected to high-temperature denaturation and then rapidly placed on ice to obtain denatured single-stranded DNA products. Connect the other end of the sequencing adapter to the single-stranded DNA product. The products after ligation of sequencing adapters were subjected to library amplification and product purification.

9. The method for constructing a dual-omics library according to claim 1, characterized in that, Constructing a single-cell transcriptome library based on the cDNA includes: The cDNA was subjected to quality testing, fragmentation, adapter ligation, library amplification, and purification.

10. The method for constructing a dual-omics library according to claim 1, characterized in that, Also includes: The concentrations of the single-cell whole-genome methylation library and the single-cell transcriptome library were determined using a Qubit fluorescence quantitative quantitation system. The distribution of nucleic acid fragments in the library was detected and quality controlled using a 4200 bioanalyzer. The single-cell whole-genome methylation library and the single-cell transcriptome library were subjected to PE150 sequencing and the sequencing results were analyzed.

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