WO2025194453A1 - Method for constructing mtsccat-seq sequencing library on the basis of droplet microfluidics - Google Patents

Abstract

Provided is a method for constructing an mtscCAT-seq sequencing library on the basis of droplet microfluidics, the method comprising: fixing and permeabilizing a cell by using a fixative and a lysing agent, respectively; use a transposase to treat the cell which has been subjected to fixation and permeabilization, which transposase is embedded with a first specific tag sequence, so as to obtain gDNA and mtDNA which are linked to the first specific tag sequence, the gDNA being derived from an open chromatin region; treating the cell by using a transcriptome capture sequence, which transcriptome capture sequence comprises a second specific tag sequence, so as to obtain, by means of reverse transcription, cDNA linked to the second specific tag sequence; generating a droplet on the basis of droplet microfluidics so as to encapsulate the cell and a first microbead within the droplet, wherein the gDNA, the mtDNA and the cDNA are captured by the first microbead; and obtaining a sequencing library for mtscCAT-seq on the basis of the captured gDNA, mtDNA and cDNA.

Description

Construction of mtscCAT-seq sequencing library based on droplet microfluidics Technical Field

The present application relates to the field of single-cell sequencing technology, and specifically to a sequencing library construction method for a sequencing technology (mitochondrial single-cell chromatin accessibility and transcriptome sequencing, mtscCAT-seq) for simultaneously capturing the mitochondrial genome, chromatin open region genome, and transcriptome based on droplet microfluidics.

Background Art

With the continuous advancement of single-cell technologies, single-cell multi-omics technologies are also developing rapidly, enabling the simultaneous detection of two or more omics within the same cell. Examples include the combined detection of single-cell transcriptomes with single-cell ATAC (attributable to accessed chromatin regions), and the combined detection of single-cell transcriptomes with immune repertoires and surface proteins. The combined detection of single-cell multi-omics can provide multiple analytical perspectives and a more complete picture of the gene regulatory networks in complex tissues.

Currently, single-cell multi-omics technologies have gradually expanded into large-scale single-cell analyses. For example, the sequencing technology mtscATAC-seq, proposed by Vijay G. Sankaran's team at Harvard University, is primarily based on scATAC-seq technology from 10×Genomics to detect mitochondrial DNA (mtDNA) mutations in single cells. ASAP-seq, building on mtscATAC-seq, can simultaneously detect cell surface and intracellular proteins. DOGMA-seq, based on the multi-omics kit Multiome from 10×Genomics, can perform multimodal analysis of chromatin accessibility, gene expression, and protein in the same cell, enabling multi-omics analysis at the single-cell level. However, these technologies are associated with high experimental costs, cumbersome and time-consuming experimental procedures, low capture efficiency, and the subsequent amplification of multi-omics products is prone to nonspecific amplification. Furthermore, robust sample processing protocols suitable for large-scale experiments targeting immune cells from a study population, such as peripheral blood mononuclear cells (PBMCs), have yet to be published.

Therefore, there is an urgent need to provide a single-cell transcriptome and single-cell epigenome combined detection technology that does not rely on multi-omics technologies in related technologies, can directly realize biological samples (especially immune cell samples that are difficult to process, etc.), and can simultaneously detect mtDNA mutations.

Summary of the Invention

To this end, an embodiment of the present application provides a mtscCAT-seq based on droplet microfluidics.

In a first aspect, an embodiment of the present application proposes a method for constructing a sequencing library for mtscCAT-seq based on droplet microfluidics, comprising: fixing and permeabilizing cells using a fixative and a lysing agent, respectively; treating the fixed and permeabilized cells using a transposase, wherein the transposase is embedded with a first specific tag sequence, to obtain gDNA and mtDNA connected to the first specific tag sequence, and the gDNA is derived from an open chromatin region; treating the cells using a transcriptome capture sequence, wherein the transcriptome capture sequence contains a second specific tag sequence, to obtain cDNA connected to the second specific tag sequence by reverse transcription; generating droplets based on droplet microfluidics to enclose the cells and first microbeads in the droplets, wherein the gDNA, the mtDNA and the cDNA are captured by the first microbeads; and obtaining a sequencing library for mtscCAT-seq based on the captured gDNA, mtDNA and cDNA.

In some embodiments, the fixative is formaldehyde. In some embodiments, the fixative is formaldehyde with a final concentration of 0.1%-0.5%, preferably with a final concentration of 0.1%.

In some embodiments, the cleavage agent is ethylphenyl polyethylene glycol (NP40) and/or an NP40 substitute, wherein the NP40 substitute is octylphenoxy poly(ethyleneoxy)ethanol, branched. In some embodiments, the cleavage agent is NP40 and/or an NP40 substitute at a final concentration of 0.1%.

In some embodiments, a microbead tag sequence is connected to the surface of the first microbead, and the microbead tag sequence binds to the first specific tag sequence of the gDNA and the mtDNA and the second specific tag sequence of the cDNA, respectively, to capture the gDNA, mtDNA and cDNA, wherein the microbead tag sequence is microbead specific.

In some embodiments, after the gDNA, mtDNA, and cDNA are captured by the first microbeads, the method further comprises: performing in-droplet amplification on the gDNA, mtDNA, and cDNA based on the binding of the microbead tag sequence of the first microbead to the gDNA, mtDNA, and cDNA to obtain amplification products, wherein the amplification products include gDNA and mtDNA simultaneously connected to the microbead tag sequence and the first specific tag sequence, and cDNA simultaneously connected to the microbead tag sequence and the second specific tag sequence.

In some embodiments, after performing the in-droplet amplification, the method further comprises: disrupting the droplet to release the amplification product. In some embodiments, the method further comprises purifying the amplification product. In some embodiments, the amplification product is purified using a second microbead, which may be a silica-based microbead.

In some embodiments, the method further comprises: separating the cDNA from the gDNA and mtDNA using a third microbead, wherein the surface of the third microbead is connected to an attachment molecule, the cDNA contains an attachment molecule affinity sequence, and the attachment molecule specifically binds to the attachment molecule affinity sequence to separate the cDNA from the gDNA and mtDNA.

In some embodiments, the attachment molecule is streptavidin, and the attachment molecule affinity sequence is a biotin sequence. In some embodiments, the biotin sequence is contained within the second specific tag sequence of the cDNA.

In some embodiments, the method further comprises: performing an out-of-droplet amplification on the gDNA, mtDNA, and cDNA.

In some embodiments, obtaining a sequencing library for mtscCAT-seq based on the captured gDNA, mtDNA, and cDNA specifically includes: constructing a library for the captured gDNA, mtDNA, and cDNA to obtain the sequencing library for mtscCAT-seq.

In some embodiments, the method further comprises: shearing the cDNA, and constructing a sequencing library based on the sheared cDNA.

In some embodiments, the transposase is Tn5 transposase.

In some embodiments, the first specific tag sequence includes a first specific tag sequence a and a first specific tag sequence b. Optionally, the first specific tag sequence a is as shown in SEQ ID NO: 1, and the first specific tag sequence b is as shown in SEQ ID NO: 2.

In some embodiments, the transcriptome capture sequence comprises polythymidine nucleotides (poly T) for capturing mRNA in the cell. Optionally, the transcriptome capture sequence is as shown in SEQ ID NO: 3.

In some embodiments, the second specific tag sequence comprises a unique molecular identifier (UMI).

In some embodiments, the microbead tag sequence comprises sequences i and ii, wherein sequence i binds to all or part of the first specific tag sequence and the second specific tag sequence to capture the gDNA, mtDNA, and cDNA. In some embodiments, sequence ii comprises a microbead-specific microbead-specific tag sequence. In some embodiments, sequence ii further comprises a linker sequence.

In some embodiments, obtaining a sequencing library for mtscCAT-seq based on the captured gDNA, mtDNA, and cDNA further comprises: preparing DNA nanoballs by rolling circle amplification based on the captured gDNA, mtDNA, and cDNA to obtain a sequencing library for mtscCAT-seq.

In some embodiments, the cells are mammalian cells, preferably human cells, and the mammalian cells are derived from one or more cells in body fluids, body excretions, body secretions, lymphoid tissue, tonsils, bone marrow, muscle, liver, spleen, kidney, lung, heart, brain, intestine, stomach, pancreas, thymus, bladder, or skin. In some embodiments, the cells are one or more cells in PBMC, tonsil cells, lymphocytes, bone marrow cells, spleen cells, and thymocytes.

The present application also provides a droplet microfluidics-based mtscCAT-seq method, comprising: constructing a sequencing library according to the method for constructing a sequencing library for droplet microfluidics-based mtscCAT-seq described in any of the above embodiments; and sequencing the library. In some embodiments, the sequencing is second-generation sequencing and/or third-generation sequencing. In some embodiments, the sequencing is based on DNA nanoballs.

The embodiments of the present application also propose a single-cell multi-omics analysis method based on mtscCAT-seq, comprising: performing mtscCAT-seq according to the droplet microfluidics-based mtscCAT-seq method described in any of the above embodiments to obtain mtscCAT-seq data; and analyzing the mtscCAT-seq data to obtain comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome and mitochondrial mutations based on the microbead label sequences in the mtscCAT-seq data.

In some embodiments, comprehensive multi-omic information on single-cell chromatin accessibility, transcriptome, and mitochondrial mutations is obtained based on the bead tag sequence in the mtscCAT-seq data, including: distinguishing gDNA, mtDNA, and cDNA originating from the same cell based on the bead tag sequence of the first bead, wherein gDNA, mtDNA, and cDNA containing the same bead tag sequence are determined to originate from the same droplet and the same cell, and gDNA, mtDNA, and cDNA containing different bead tag sequences are determined to originate from different droplets and different cells; and analyzing the gDNA, mtDNA, and cDNA originating from the same cell to obtain the comprehensive multi-omic information on single-cell chromatin accessibility, transcriptome, and mitochondrial mutations.

The present application also proposes a high-throughput single-cell multi-omics analysis method based on mtscCAT-seq, comprising: constructing a sequencing library according to the sequencing library construction method for mtscCAT-seq based on droplet microfluidics described in any of the above embodiments, wherein m cells and n first microbeads are enclosed in a single droplet, wherein m≤1 and n≥1, preferably 1≤n≤4; sequencing the library to obtain mtscCAT-seq data; and analyzing the mtscCAT-seq data to obtain a specific tag sequence, a second specific tag sequence and a microbead tag sequence according to the mtscCAT-seq data. Comprehensive multi-omics information on chromatin accessibility, transcriptome, and mitochondrial mutations in single cells.

In some embodiments, comprehensive multi-omic information on single-cell chromatin accessibility, transcriptome, and mitochondrial mutations is obtained based on the first specific tag sequence, the second specific tag sequence, and the microbead tag sequence in the mtscCAT-seq data, including: distinguishing gDNA, mtDNA, and cDNA originating from the same droplet based on the microbead tag sequence; determining gDNA, mtDNA, and cDNA originating from the same cell in the same droplet based on the first specific tag sequence and the second specific tag sequence; and analyzing the gDNA, mtDNA, and cDNA originating from the same cell to obtain the comprehensive multi-omic information on single-cell chromatin accessibility, transcriptome, and mitochondrial mutations.

The embodiments of the present application also propose a single-cell multi-omics combined analysis method, including: performing transcriptomics and/or epigenomics and/or mitochondrial mutation analysis according to the single-cell multi-omics analysis method based on mtscCAT-seq described in any of the above embodiments or the high-throughput single-cell multi-omics analysis method based on mtscCAT-seq described in any of the above embodiments; and performing a combined analysis based on genomics, proteomics and/or metabolomics.

In some embodiments, the epigenomics includes chromatin accessibility and one or more of: histone modifications, DNA methylation, RNA methylation, and non-coding RNA.

The technical solution of this application achieves the following technical effects:

The mtscCAT-seq library construction and sequencing method in the embodiment of the present application can directly realize the joint detection of single-cell transcriptome and single-cell epigenome through special experimental design and multiple tag joint labeling, thereby reducing the time-consuming overall experimental process, while retaining mitochondrial genome information for related research, effectively improving the sequence capture efficiency of single-cell transcriptomics, epigenomics (chromatin accessibility) and mitochondrial omics, and obtaining a library with higher specificity. Based on this high-specificity library, combined with sequencing and data analysis, a more complete single-cell atlas is provided, and mitochondrial genome information is effectively retained, providing a detection technology platform for studying mitochondrial mutations related to various clinical phenotypes, and providing a basis for the multi-omics joint analysis of single cells.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

FIG1 is a method for constructing a sequencing library for mtscCAT-seq based on droplet microfluidics according to an embodiment of the present application;

FIG2 is an overall flow chart of the mtscCAT-seq method based on droplet microfluidics according to an embodiment of the present application;

FIG3 is a diagram showing a chip according to an embodiment of the present application;

FIG4 is a schematic diagram of quality control of transcriptome sequencing results according to Example 1 of the present application;

FIG5 is a schematic diagram of quality control of epigenomic sequencing results according to Example 1 of the present application;

FIG6 is a transcriptome cluster diagram according to Example 1 of the present application;

FIG7 shows the transcriptome clustering effect of two samples according to Example 1 of the present application;

FIG8 is a cluster diagram of the apparent groups according to Example 1 of the present application;

FIG9 shows the apparent group clustering effect of two samples according to Example 1 of the present application.

DETAILED DESCRIPTION

The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

This application is made based on the following knowledge of the inventors:

Among the related technologies, those that can be used to detect single-cell multi-omics include: mtscATAC-seq (Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling | Nature Biotechnology) developed by Vijay G. Sankaran's team at Harvard University based on 10×Genomics' technology, and ASAP-seq (Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells | Nature Biotechnology) based on mtscATAC-seq. e Biotechnology), DOGMA-seq based on 10×Genomics’ multi-omics kit Multiome (Comprehensive benchmarking of CITE-seq versus DOGMA-seq single cell multimodal omics - PubMed (nih.gov)), and 10×Genomics single cell multimodal ATAC & gene expression technology (https://cdn.10xgenomics.com/image/upload/v1666737555/support-documents/CG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevF.pdf).

However, these single-cell multi-omics detection technologies have many defects, such as high experimental costs, cumbersome experimental processes, long time consumption, low capture efficiency, and prone to non-specific amplification when amplifying multi-omics products separately in the later stage. In addition, ASAP-seq needs to be combined with CITE-seq to perform integrated analysis of single-cell transcriptomic information, making the experiment more complicated and more expensive. As for the 10×Genomics single-cell multi-omics ATAC&gene expression technology, it is necessary to complete reverse transcription and capture of target sequences in droplets, and use multiple sequences to capture single-cell ATAC and single-cell RNA fragments respectively. This strategy of performing reverse transcription and sequence capture in the same step makes the overall experimental process time-consuming; at the same time, the subsequent amplification process of products for different omics is prone to non-specific amplification, which reduces the specificity of the library, thereby affecting the accuracy of the analysis results.

In addition, in related technologies, robust sample processing experimental procedures suitable for large-scale experiments for immune cells of the research population, such as peripheral blood mononuclear cells (PBMCs), have not yet been published, which limits the large-scale multi-omics analysis process of these cells.

After many experiments and tests, the inventors of this application have developed a method for constructing sequencing libraries for mtscCAT-seq based on droplet microfluidics. This method is based on a droplet microfluidics platform. Through special experimental design and multi-label joint labeling, it can directly realize the joint detection of single-cell transcriptome and single-cell epigenome, while retaining mitochondrial genome information for related research. The method proposed in the examples of this application has a simple overall experimental process, short time consumption, and high capture efficiency. In addition, by separately amplifying the products of different omics, it effectively reduces the interference of non-specific amplification and improves the specificity of the library. This method can effectively improve the accuracy of the analysis results; at the same time, the method can capture and produce multi-omics data with high throughput. The output data can identify more cell subpopulations, providing a feasible basis for high-throughput research of single-cell multi-omics.

It is understood that the methods proposed in the embodiments of the present application can be used for conventional biological samples, such as one or more cells from body fluids, body excretions, body secretions, lymphoid tissue, tonsils, bone marrow, muscle, liver, spleen, kidney, lung, heart, brain, intestine, stomach, pancreas, thymus, bladder, and skin. In addition, the methods of the embodiments of the present application are particularly suitable for samples that are difficult to process, such as immune cells (e.g., one or more cells in PBMCs, tonsil cells, lymphocytes, bone marrow cells, splenocytes, and thymocytes), and can obtain high-quality single-cell libraries, thereby providing a stable and efficient experimental process for cell types that are difficult to process in traditional technologies.

In the embodiments of the present application, "open chromatin region" or "accessible-chromatin" refers to the exposed DNA (region) presented by opening the compact chromatin structure during DNA replication and transcription. "Chromatin accessibility" refers to the property of the exposed DNA (i.e., open chromatin region/genomic region of open chromatin) presented by the opening of the compact chromatin structure, which allows the binding of regulatory factors. In the embodiments of the present application, the study of chromatin accessibility can be used to analyze the region where transcription occurs, the regulation of transcription factors, and the retrieval of motif-corresponding transcription factors, thereby revealing gene expression regulatory information.

In the examples of the present application, "mtscCAT-seq (mitochondrial single-cell chromatin accessibility and transcriptome sequencing)" refers to the joint sequencing of chromatin accessibility regions (i.e., gDNA), mitochondrial genome (mtDNA), and transcriptome sequences (especially mRNA) in single cells. Through the correlation analysis of the three, the expression and regulatory information of genes can be more accurately confirmed and revealed.

Figure 1 shows a method for constructing a sequencing library for mtscCAT-seq based on droplet microfluidics according to an embodiment of the present application. As shown in Figure 1 , the method may include steps S101-S105.

S101: Fix and permeabilize cells using fixative and lysis reagents, respectively.

In the embodiments of the present application, the fixative can be formaldehyde, optionally with a final concentration of 0.1%-0.5% (w/v). In some embodiments, the fixative is formaldehyde with a final concentration of 0.1%. The embodiments of the present application use a specific concentration of fixative to cross-link and fix the cells, thereby preserving the original chromatin landscape of the cells and the mitochondrial genome to the greatest extent, providing a stable foundation for subsequent multi-omics analysis including mitochondrial genomics.

In the embodiments of the present application, the cleavage agent can be octylphenoxypolyethoxyethanol (NP40, C ₁₅ H ₂₄ O(C ₂ H ₄ O) _n ) and/or an NP40 substitute, optionally NP40 and/or an NP40 substitute at a final concentration of 0.1% (w/v). In some embodiments, the NP40 substitute is branched octylphenoxy poly(ethyleneoxy)ethanol (C ₂ H ₄ O) _n C ₁₄ H ₂₂ O). In some embodiments, the cleavage agent can be formulated into a lysis buffer, for example, by combining the cleavage agent at the above-mentioned specific concentrations with one or more components selected from the group consisting of Tris (optionally pH 8.0)/Tris-HCl (optionally pH 7.4), Sucrose, KCl/NaCl, MgCl 2 , DTT, protease inhibitor cocktail, RNase inhibitor, and 1% BSA to prepare a lysis buffer. These components can provide reaction conditions for the cleavage agent. The specific concentration of the fixative lysing agent proposed in the embodiment of the present application is simple in composition and can gently lyse the The cells are lysed and permeabilized to effectively preserve the mitochondrial genome, thereby enabling efficient detection of mitochondrial mutations.

It can be understood that the method proposed in the embodiment of the present application combines optimized fixation and lysis parameters, based on which mitochondrial DNA can be efficiently and stably captured, and the constructed library is of better quality, which is conducive to the production of stable multi-omics data and is also conducive to the detection of mutations in the mitochondrial genome.

In the examples of the present application, after cell fixation and before permeabilization, the method further includes: terminating the fixation and crosslinking using a terminator. In some embodiments, the terminator can be glycine, for example, using a glycine solution with a final concentration of 0.125M to 0.25M to terminate the reaction. It is understood that other concentrations of glycine or other terminators can also be used, and this application is not limited thereto.

S102: treating the fixed and permeabilized cells with a transposase, wherein the transposase is embedded with a first specific tag sequence, to obtain gDNA and mtDNA connected to the first specific tag sequence, wherein the gDNA is derived from the open chromatin region.

In the embodiments of the present application, "treating cells with a transposase" refers to attacking permeabilized cells with a transposase embedded with a specific tag sequence (first specific tag sequence). The transposase can randomly bind to and cut the DNA in the open chromatin region and the fixed naked mtDNA, and can simultaneously insert its pre-embedded first specific tag sequence at the cutting site to obtain mtDNA connected to the first specific tag sequence and gDNA derived from the open chromatin region.

In an embodiment of the present application, the first specific tag sequence may include a first specific tag sequence a and a first specific tag sequence b, that is, the first specific tag sequence a and the first specific tag sequence b are respectively embedded on both sides of the transposase. In some embodiments, the first specific tag sequence a and/or the first specific tag sequence b are specific sequences, and all or part of the sequence of the first specific tag sequence a and/or the first specific tag sequence b is bound to the microbead tag sequence attached to the first microbead to capture the mtDNA connected to the first specific tag sequence and the gDNA derived from the open region of chromatin; subsequently, the mtDNA and gDNA derived from the same single cell are distinguished by the microbead tag sequence. In some embodiments, the first specific tag sequence a is as shown in SEQ ID NO: 1, and the first specific tag sequence b is as shown in SEQ ID NO: 2.

In the embodiments of the present application, the first specific tag sequence may further include a known sequence portion for subsequent amplification, purification, library construction, and sequencing, etc., and the known sequence portion may include, but is not limited to, an adapter sequence. It should be noted that in the mtscCAT-seq method proposed in the embodiments of the present application, the length and specific sequence of the known sequence in the first specific tag sequence can be determined as needed, as long as high-throughput single-cell sequencing can be ensured. This application does not impose any restrictions on the length and specific sequence of the known sequence.

In some embodiments, the transposase can be Tn5 transposase.

S103: treating the cell with a transcriptome capture sequence, wherein the transcriptome capture sequence comprises a second specific tag sequence, so as to obtain cDNA connected to the second specific tag sequence by reverse transcription.

In the present application, "treating cells with transcriptome capture sequences" means that the transcriptome capture sequences enter the nucleus of permeabilized cells, and the transcriptome capture sequences contain polythymidine nucleotides (poly T), which interact with the cells through the poly T sequences. mRNA in the nucleus to capture the transcriptome in single cells.

In an embodiment of the present application, the transcriptome capture sequence may include a second specific tag sequence. In some embodiments, the second specific tag sequence includes or is a unique molecular identifier (UMI). It is understandable that by using the transcriptome capture sequence to capture mRNA and perform reverse transcription, the second specific tag sequence (such as UMI) can be labeled with each mRNA of each single cell, and the UMIs connected to each mRNA are different from each other. It is understandable that by introducing UMI to each cDNA chain through reverse transcription, the products amplified from the same cDNA in the subsequent library construction process all have the same tag, while the natural repeat fragments have different tags. Later, by using UMI to filter the data, the cDNA in the sample can be accurately counted, thereby distinguishing between multiple copies of the sequence and false multiple copies (caused by PCR preference or other factors), thereby obtaining more accurate transcriptome information.

In the examples of this application, the transcriptome capture sequence may also include a known sequence portion for subsequent amplification, binding to the microbead tag sequence, separation of gDNA and mtDNA, purification, library construction, and sequencing. This known sequence portion includes, but is not limited to, a complementary sequence to the microbead tag sequence, a linker sequence, and an affinity sequence for an attachment molecule (e.g., a biotin sequence). It should be noted that in the mtscCAT-seq method proposed in the examples of this application, the length and specific sequence of the known sequence in the second specific tag sequence can be determined as needed, as long as single-cell sequencing can be guaranteed. This application does not impose any restrictions on the length and specific sequence of this known sequence.

In an embodiment of the present application, the transcriptome capture sequence can be shown as SEQ ID NO: 3.

The mtscCAT-seq proposed in the examples of the present application completes reverse transcription before droplet generation. Compared with in-droplet reverse transcription in traditional technologies, in situ reverse transcription is performed after the cells are in situ cross-linked and fixed. This effectively avoids mRNA degradation during cell phase preparation and droplet sealing, thereby improving the mRNA capture efficiency and achieving efficient cDNA labeling.

S104: Generate droplets based on droplet microfluidics to enclose the cells and first microbeads in the droplets, wherein the gDNA, the mtDNA, and the cDNA are captured by the first microbeads.

In the embodiments of the present application, "droplet microfluidics" refers to a technology that operates on a microfluidic platform to operate on a microbead phase, a cell phase, and an oil phase to generate droplets based on the oil phase to enclose the microbead phase and the cell phase in the droplets. In some embodiments, the droplets are simultaneously enclosed with a first microbead and a cell labeled with a first specific tag sequence and a second specific tag sequence. The surface of the first microbead is connected to a microbead tag sequence, and the microbead tag sequence comprises sequences i and ii, wherein sequence i binds to the first specific tag sequence of gDNA and mtDNA and the second specific tag sequence of cDNA in the cell, respectively, to capture the gDNA, mtDNA, and cDNA; sequence ii comprises a microbead-specific microbead-specific tag sequence, that is, the microbead tag sequences (i.e., sequence ii) attached to the surface of each microbead are different from each other. It can be understood that the labeled gDNA, mtDNA, and cDNA in the cells are captured by microbeads in the droplets, and based on the binding of the sequence i of the microbead tag sequence of the first microbead to the gDNA, mtDNA, and cDNA, the gDNA, mtDNA, and cDNA are amplified in the droplet to obtain amplification products, wherein the amplification products include gDNA and mtDNA simultaneously linked to the microbead tag sequence and the first specific tag sequence, and cDNA simultaneously linked to the microbead tag sequence and the second specific tag sequence. This provides the same microbead label sequence for gDNA, mtDNA, and cDNA in the same droplet, thereby tracing the source of the same droplet, or the same single cell, in subsequent data analysis.

In the embodiments of the present application, the microbead tag sequence (e.g., sequence ii thereof) may also include a known sequence portion for subsequent amplification, purification, library construction, and sequencing, etc., and the known sequence portion includes, but is not limited to, an adapter sequence. It should be noted that in the mtscCAT-seq proposed in the embodiments of the present application, the length and specific sequence of the known sequence in the microbead tag sequence can be determined as needed, as long as single-cell sequencing can be guaranteed. This application does not impose any restrictions on the length and specific sequence of the known sequence.

In the embodiment of the present application, the first microbeads may be magnetic beads or gel beads.

The method proposed in the embodiment of the present application can achieve the simultaneous capture of gDNA, mtDNA and cDNA using only one microbead label sequence through the ingenious design of the label sequence, thereby greatly simplifying the experimental operation, reducing the time consumption of the overall experimental process and improving the capture efficiency.

S105: Sequencing library for mtscCAT-seq is obtained based on the captured gDNA, mtDNA, and cDNA.

In the examples of the present application, based on the captured gDNA, mtDNA connected to a first specific tag sequence and a microbead tag sequence, and cDNA connected to a second specific tag sequence and a microbead tag sequence, a sequencing library for mtscCAT-seq can be obtained by destroying the droplets containing them and optionally performing cDNA shearing, amplification, linker addition, and purification.

In some embodiments, the droplets are disrupted to release the amplified products. In some embodiments, the droplets can be disrupted by emulsifying the droplets (e.g., using a breakage buffer) to release the amplified products within the droplets. It is understood that this breakage step can be performed on individual droplets, or all droplets can be collected and then processed together. As long as the oil phase used to form the droplet envelope is disrupted, the processing is not limited. In some embodiments, the breakage buffer is an emulsifier, etc.

In some embodiments, the method further comprises purifying the amplified product, for example, using a second microbead to purify the amplified product, to obtain purified gDNA, mtDNA, and cDNA. In some embodiments, the second microbead can be a silica-based microbead, such as a silica-based magnetic bead. It is understood that the second microbead has a specific affinity for DNA molecules, thereby achieving purification of gDNA, mtDNA, and cDNA by specifically capturing these DNA molecules.

In an embodiment of the present application, the method further comprises: separating the cDNA from the gDNA and mtDNA using a third microbead, wherein the third microbead has an attachment molecule attached to its surface, the cDNA contains an attachment molecule affinity sequence, and the attachment molecule specifically binds to the attachment molecule affinity sequence to separate the cDNA from the gDNA and mtDNA. In some embodiments, the attachment molecule is streptavidin, and the attachment molecule affinity sequence is a biotin sequence.

In some embodiments, the biotin sequence is contained within the second specific tag sequence of the cDNA. In other embodiments, when the method further comprises synthesizing a second strand of cDNA, the biotin sequence may also be contained within the primers used to amplify the cDNA, so as to introduce the biotin sequence into the cDNA via the in-droplet amplification. The biotin sequence is preferably located at the 5' end of the primer.

In the examples of the present application, cDNA can be separated from gDNA and mtDNA after droplet destruction and before amplification outside the droplet, and the separated cDNA, gDNA, and mtDNA are subsequently amplified and library constructed separately. The method proposed in the examples of the present application separates the products of the two omics before amplification and amplifies them separately afterwards, thereby avoiding cross-contamination of multi-omics products and potential nonspecific amplification, and the resulting library is more specific.

In an embodiment of the present application, the method further comprises: performing extra-droplet amplification on the gDNA, mtDNA, and cDNA. Preferably, the separated gDNA, mtDNA, and cDNA are separately subjected to extra-droplet amplification. By separating the products of the two omics prior to amplification and subsequently amplifying them separately, the embodiment of the present application avoids cross-contamination of multi-omics products and potential nonspecific amplification, thereby resulting in a more specific library.

In the embodiment of the present application, step S105 also includes: preparing DNA nanoballs by rolling circle amplification based on the captured gDNA, mtDNA and cDNA to obtain a sequencing library for mtscCAT-seq. It is understood that the mtscCAT-seq of the embodiment of the present application can also prepare DNA nanoballs (DNBs) by rolling circle amplification, and use the DNBs as sequencing libraries. It is understood that by further preparing gDNA, mtDNA and cDNA into DNBs, the sequencing signal intensity can be effectively enhanced and the sequencing accuracy can be improved, thereby achieving high-precision, high-quality sequencing.

In the embodiments of the present application, the cells may be mammalian cells or non-mammalian cells, preferably human cells, and the mammalian cells may be derived from one or more cells in body fluids, body excretions, body secretions, lymphoid tissue, tonsils, bone marrow, muscles, liver, spleen, kidney, lung, heart, brain, intestine, stomach, pancreas, thymus, bladder, or skin. In some embodiments, the cells are PBMCs and/or immune organ cells, such as one or more cells in tonsil cells, lymphocytes, bone marrow cells, spleen cells, and thymocytes. The mtscCAT-seq proposed in the embodiments of the present application can efficiently process PBMCs and immune organ cells that are difficult to process in traditional technologies, and by using optimized milder fixation, lysis, and permeabilization conditions, it effectively retains the mitochondrial genome and more efficiently captures the mitochondrial genome. The mtscCAT-seq proposed in the embodiments of the present application has been systematically optimized for sample processing of PBMCs and immune organ cells, providing them with a robust experimental process for obtaining high-quality single-cell libraries.

The library construction method of mtscCAT-seq proposed in the embodiment of the present application, through special experimental design and multi-label joint labeling, can directly realize the joint detection of single-cell transcriptome and single-cell ATAC, while retaining mitochondrial genome information for related research. The overall experimental process of the method proposed in the embodiment of the present application is simple, time-consuming, and has a high capture efficiency, and by amplifying the products of different groups separately, the interference of non-specific amplification is effectively reduced, the library specificity is improved, thereby effectively improving the accuracy of the analysis results; at the same time, the method can capture and output multi-omics data with high throughput, and the output data can identify more cell subpopulations, providing a feasibility basis for high-throughput research on single-cell multi-omics. In addition, the method of the embodiment of the present application can also be applied to samples that are difficult to handle, such as PBMC and immune organ samples, and high-quality single-cell libraries can be obtained, thereby providing a set of stable and efficient experimental processes for cell types that are more difficult to handle in traditional technologies.

A second embodiment of the present application provides a mtscCAT-seq method based on droplet microfluidics, comprising: constructing a sequencing library according to the sequencing library construction method for mtscCAT-seq based on droplet microfluidics described in any of the above embodiments; and sequencing the library. In some embodiments, the sequencing is second-generation sequencing and/or third-generation sequencing. In some embodiments, the sequencing is based on DNA nanoballs. It is understood that the construction and sequencing of libraries for gDNA, mtDNA, and cDNA in the methods of the embodiments of the present application can be performed with reference to the standard procedures of the sequencing platform used, without limitation herein.

The droplet microfluidics-based mtscCAT-seq method proposed in the embodiments of the present application sequences gDNA, mtDNA, and cDNA, and determines the gDNA, mtDNA, and cDNA originating from the same cell based on the microbead tag sequence in the sequencing data (wherein the gDNA, mtDNA, and cDNA originating from the same cell are connected to the same microbead tag sequence), and can further perform specific analysis of the transcriptome based on a second specific tag sequence, as well as joint analysis of multiple omics such as gDNA, mtDNA, and cDNA, thereby obtaining highly accurate single-cell multi-omics information.

FIG2 is a flow chart of the overall mtscCAT-seq method based on droplet microfluidics according to a specific embodiment of the present application. As shown in FIG2 , the mtscCAT-seq method proposed in the embodiment of the present application may include:

a. After fixing cells with 0.1% formaldehyde, permeabilize the cell membrane with a mild lysis buffer containing only 0.1% NP40 to preserve mitochondrial DNA.

b. Using Tn5 transposase pre-embedded with a specific tag sequence (i.e., a first specific tag sequence), the cells are transposed in situ to obtain DNA fragments in the open chromatin region with the specific tag sequence (i.e., gDNA fragments) and mitochondrial genome fragments with the specific tag sequence (i.e., mtDNA fragments);

c. After in situ transposition, a poly T primer (i.e., transcriptome capture sequence) containing a specific tag sequence (i.e., a second specific tag sequence) is used to capture the mRNA sequence with a poly A tail in the nucleus, and then reverse transcribe it into cDNA. Optionally, this step can also include second-strand synthesis of the cDNA.

d. Separately preparing a magnetic bead phase (i.e., first microbeads) and a cell phase, generating droplets using a droplet microfluidics device and performing PCR pre-amplification (i.e., in-droplet pre-amplification), wherein the first specific tag sequence attached to the gDNA and mtDNA and the second specific tag sequence attached to the cDNA are captured by the microbead tag sequence attached to the magnetic beads, and the PCR pre-amplification is performed based on the binding of the first specific tag sequence and the second specific tag sequence attached to the cDNA;

e. The amplified droplets are demulsified and purified. During this process, streptavidin magnetic beads (i.e., third beads) are used to specifically capture the cDNA with biotin sequences to achieve the purpose of separating it from gDNA and mtDNA. After separation, they are amplified again (i.e., off-droplet amplification), and libraries are constructed and sequenced.

Therefore, the overall technical route of the mtscCAT-seq method proposed in the embodiment of the present application is as follows: 0.1% formaldehyde fixation of cells → 0.1% NP40 lysis solution permeabilization of cell membranes → in situ transposition of cells (based on Tn5 transposase embedded with a specific tag sequence) → in situ reverse transcription of the nucleus (based on polyT primers containing a specific tag sequence) → droplet generation and PCR pre-amplification (in-droplet amplification) → emulsion breaking and purification → cDNA separation with streptavidin magnetic beads → cDNA, gDNA, and mtDNA are amplified separately (out-of-droplet amplification) → library construction → sequencing. The mtscCAT-seq method proposed in the embodiment of the present application comprehensively optimizes the fixation and lysis permeabilization of cells, so that it can be used to process various types of cells, especially special cells such as PBMC and immune organ cells that are not efficiently processed by traditional technologies, while retaining the mitochondrial genome more completely; at the same time, this method completes the reverse transcription of the transcriptome before droplet generation, thereby avoiding the degradation of mRNA in subsequent processes to the greatest extent, thereby improving The overall stability of the experiment is improved; in addition, this method can complete the simultaneous capture of gDNA, mtDNA and cDNA using only one microbead label sequence, thereby greatly simplifying the experimental operation, reducing the overall experimental process time and improving the capture efficiency.

The third aspect of the present application also proposes a single-cell multi-omics analysis method based on mtscCAT-seq, comprising: performing mtscCAT-seq according to the droplet microfluidics-based mtscCAT-seq method described in any of the above embodiments to obtain mtscCAT-seq data; and analyzing the mtscCAT-seq data to obtain comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome and mitochondrial mutations based on the microbead label sequence in the mtscCAT-seq data.

In an embodiment of the present application, comprehensive multi-omic information on single-cell chromatin accessibility, transcriptome, and mitochondrial mutations is obtained based on the microbead label sequence in the mtscCAT-seq data, including: distinguishing gDNA, mtDNA, and cDNA originating from the same cell based on the microbead label sequence of the first microbead, wherein gDNA, mtDNA, and cDNA containing the same microbead label sequence are determined to originate from the same droplet and the same cell, and gDNA, mtDNA, and cDNA containing different microbead label sequences are determined to originate from different droplets and different cells; and analyzing the gDNA, mtDNA, and cDNA originating from the same cell to obtain the comprehensive multi-omic information on single-cell chromatin accessibility, transcriptome, and mitochondrial mutations.

The third aspect of the embodiments of the present application also proposes a high-throughput single-cell multi-omics analysis method based on mtscCAT-seq, comprising: constructing a sequencing library according to the sequencing library construction method for droplet microfluidics-based mtscCAT-seq described in any of the above embodiments, wherein m cells and n first microbeads are enclosed in a single droplet, where m≤1 and n≥1, preferably 1≤n≤4; sequencing the library to obtain mtscCAT-seq data; and analyzing the mtscCAT-seq data to obtain comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome and mitochondrial mutations based on the first specific tag sequence, the second specific tag sequence and the microbead tag sequence in the mtscCAT-seq data.

In an embodiment of the present application, the comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome and mitochondrial mutations is obtained according to the first specific tag sequence, the second specific tag sequence and the microbead tag sequence in the mtscCAT-seq data, including: distinguishing gDNA, mtDNA and cDNA originating from the same droplet according to the microbead tag sequence; determining gDNA, mtDNA and cDNA originating from the same cell in the same droplet according to the first specific tag sequence and the second specific tag sequence; and analyzing the gDNA, mtDNA and cDNA originating from the same cell to obtain the comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome and mitochondrial mutations. It can be understood that by multiple labeling of the microbead tag sequence, the first specific tag sequence and the second specific tag sequence, it will be possible to effectively distinguish the droplet origin and cellular origin of gDNA, mtDNA and cDNA, thereby realizing a high-throughput single-cell multi-omics analysis method based on mtscCAT-seq.

The embodiments of the present application also propose a single-cell multi-omics combined analysis method, including: performing transcriptomics and/or epigenomics and/or mitochondrial mutation analysis according to the single-cell multi-omics analysis method based on mtscCAT-seq described in any of the above embodiments or the high-throughput single-cell multi-omics analysis method based on mtscCAT-seq described in any of the above embodiments; and performing a combined analysis based on genomics, proteomics and/or metabolomics.

In some embodiments, the epigenomics includes chromatin accessibility and one or more of: histone modifications, DNA methylation, RNA methylation and non-coding RNA.

It should be noted that the above explanation of the embodiment of the sequencing library construction method for droplet microfluidics-based mtscCAT-seq is also applicable to the droplet microfluidics-based mtscCAT-seq method, the mtscCAT-seq-based single-cell multi-omics analysis method, the mtscCAT-seq-based high-throughput single-cell multi-omics analysis method and the single-cell multi-omics combined analysis method in the embodiments of the present application, and will not be repeated here.

Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

Example 1

In this example, mtscCAT-seq was performed using PBMC samples as an example.

(1) Cryopreserved cell recovery and single cell suspension preparation (PBMC samples)

1.1 Thaw frozen cells in a 37°C water bath;

1.2 Resuspend the thawed PBMCs in 3 ml of RPMI-1640 medium supplemented with 10% FBS in a 15 ml centrifuge tube.

1.3 Mix the cells by inverting them five times, centrifuge at 300g for 5 minutes at room temperature, and remove the supernatant.

1.4 Add 500 μl of PBS supplemented with 0.04% BSA to resuspend the cell pellet;

1.5 Filter the cell suspension prepared in 1.4 through a 30 μm or 40 μm cell sieve and collect the filtered cell filtrate;

1.6 Pipette 10 μl of trypan blue dye into a centrifuge tube, add 10 μl of cell suspension, and gently pipette to mix to obtain a cell/dye mixture;

1.7 Pipette 10 μl of the cell/dye mixture into a cell counting plate. Count under a microscope. Depending on the cell concentration, pipette 500,000 cells and centrifuge at 500-1000 g, 4°C, for 3-5 min to collect the cell pellet. Set aside.

(2) Cell fixation

2.1 Prepare 0.1% formaldehyde solution according to the table below, place at room temperature, and prepare it before use.

Table 1

2.2 Prepare 0.25M glycine solution according to the table below and place it at room temperature.

Table 2

2.3 Resuspend the cell pellet from step 1.7 in 200 μl of 0.1% formaldehyde solution and incubate at room temperature for 5 min.

2.4 Add 200 μl of 0.25 M glycine solution to terminate fixation, pipette to mix, and incubate at room temperature for 5 min.

2.5 After terminating fixation, centrifuge at 1000g for 3 min at 4°C, discard the supernatant, and wash the cells twice with 200μl PBS supplemented with 0.04% BSA, and discard the supernatant.

2.6 Resuspend the cells in 40 μl of PBS supplemented with 0.04% BSA and count the cell suspension under a microscope using a cell counting plate.

(3) Magnetic bead phase preparation (Except where otherwise noted, all preparations are based on MGI's DNBelabC series high-throughput single-cell ATAC library preparation kit - droplet generation module 940-000792-00, hereinafter referred to as "940-000792-00")

3.1 Pipette 550,000 magnetic beads into a PCR tube, place it on a magnetic stand and let it stand until the liquid is clear, then remove the supernatant;

3.2 Keep the PCR tube on the magnetic rack and add 80 μl of scATAC Wash Buffer to wash the magnetic beads;

3.3 Keep the PCR tube on the magnetic stand and add the components listed in the table below to prepare the magnetic bead phase.

ad153-ISF-pho and ad153-ISR are RNA PCR amplification primers, and their sequences are: 5’-AAGCAGTGGTATCAACGCAGAGCGA-3’ (SEQ ID NO: 6) and 5’-AAGCAGTGGTATCAACGCAGAGGGG-3’ (SEQ ID NO: 7).

Table 3

(4) Cell lysis

4.1 Prepare NIM Buffer (pH 8.0) according to the following table, filter through a 0.2 μm filter, and precool at 4°C:

Table 4

4.2 Prepare Homogenization Buffer according to the following table and place on ice:

Table 5

4.3 Prepare Homogenization Buffer-washing according to the following table and place on ice:

Table 6

4.4 Transfer the 300,000 cells resuspended in 2.6 to a 1.5 ml centrifuge tube, centrifuge at 1000 g for 3 min at 4°C, and discard the supernatant.

4.5 Pipette 100 μl of pre-chilled Homogenization Buffer to resuspend the cells, pipette gently 30 times to mix, and incubate on ice for 3 min.

4.6 Add 480 μl of pre-chilled Homogenization Buffer-washing to the lysed cell suspension. Centrifuge at 1000 g for 3 min at 4°C. Discard the supernatant to wash the cells. Repeat this step once.

4.7 Resuspend the cell nuclei in 40 μl of PBS supplemented with 1% BSA and count them using a cell counting chamber and trypan blue staining.

(5) Transposition (Except where otherwise noted, all are based on MGI's DNBelabC Series High-Throughput Single-Cell ATAC Library Preparation Kit - Droplet Generation Module 940-000794-00, hereinafter referred to as "940-000794-00")

5.1 Based on the cell counting results, 200,000 cell nuclei were collected and placed in a 1.5 ml centrifuge tube. The volume was adjusted to 139 μl using PBSI (containing 1% BSA and 1 U/μl RNase inhibitor).

Add transposition buffer (40 μl of 5×TAG buffer and 21 μl of Tn5 enzyme (Neoprimaries, LS-EZ-E-00009P)) to tube 5.2. The two linker sequences that the Tn5 enzyme needs to embed are: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’ (i.e., the first specific tag sequence a, SEQ ID NO: 1); 5’-GTCTCGTGGGCTCGGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3’ (i.e., the first specific tag sequence b, SEQ ID NO: 2).

5.3 Mix the transposition system by pipetting, then divide it evenly into four 1.5 ml centrifuge tubes and transpose in a metal bath at 37°C and 500 rpm for 30 min.

5.4 After transposition is completed, combine the four tubes of transposed cells into a 1.5 ml centrifuge tube and add an equal volume (200 μl) of transposition termination buffer (prepared according to the table below). Incubate on ice for 5 minutes, centrifuge at 1000g for 3 minutes at 4°C, and discard the supernatant.

Table 7

5.5 Add 200 μl of 2 mM MgCl ₂ + PBSI, centrifuge at 1000 g for 3 min at 4°C, discard the supernatant, and resuspend the pellet with 8 μl of PBSI.

(6) Reverse transcription

6.1 Prepare the reverse transcription system (RT mix) according to the table below; divide the mixture evenly into 4 PCR tubes (keep the same number of tubes as in the transposition reaction).

Table 8

The Capture oligo primer sequence is: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNNNTTAATTAAGGVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’ (SEQ ID NO: 3, N is a specific molecular identification sequence UMI composed of random bases); the TSO primer sequence is 5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’ (SEQ ID NO: 4, where rG represents riboguanylate and +G represents fixed deoxyguanylate).

6.2 Mix the RT mix by pipetting and place it on a PCR instrument for reverse transcription reaction. The procedure is as shown in the table below.

Table 9

6.3 After reverse transcription, combine the four PCR tubes into one new PCR tube, centrifuge at 500g for 5 minutes at 4°C, and discard the supernatant.

6.4 Resuspend with appropriate amount of 1% BSA + PBS, centrifuge at 500g for 5 minutes at 4°C, and discard the supernatant.

6.5 Resuspend with appropriate amount of 1% BSA + PBS, stain with trypan blue, and then detect the cell concentration using a cell counting plate.

6.6 Take an appropriate amount of cells and prepare the cell phase (as shown in Table 10 below).

Table 10 (based on 940-000794-00)

(7) Droplet generation

7.1 Remove the surface protective film of the chip shown in Figure 3 (based on the kit 940-000148-00 module, hereinafter referred to as "940-000148-00") and place it in the chip slot area of the droplet generator.

7.2 Insert the A end of the connecting tube on the collection cover (the connecting tube that contacts the bottom of the collection tube) into the Outlet hole of the chip.

7.3 Place the 30ml syringe in the holder and adjust the plunger to the 17ml position. Use a flat needle to connect the syringe to the end of the connecting tube B on the collection tube cap (the connecting tube that does not touch the bottom of the collection tube).

7.4 Add 100 μl of droplet generation oil to the collection tube (940-000148-00), tighten the collection cap, and place the collection tube upright on the holder. Use a pipette to gently pipette and mix the cells. Add 105 μl of the cell phase prepared in step 6.6 above to the cells well of the chip, ensuring that the pipette tip touches the bottom of the well.

7.5 Use a pipette to gently pipette to mix the magnetic bead phase, and add 105 μl of the magnetic bead phase prepared in step 3.3 above to the beads well of the chip, making sure the pipette tip touches the bottom of the well.

7.6 Immediately add 350 μl of droplet generation oil to the Oil well of the chip.

7.7 Quickly pull the plunger of the syringe to the 20ml position and lock the syringe piston rod on the outer end of the base.

7.8 Start the timer for 8-10 minutes and collect the droplets.

7.9 After 8-10 minutes, immediately loosen the collection cap on the collection tube, pull out the connecting tube from the chip outlet hole, stretch the connecting tube vertically to allow the droplets in the tube to flow into the collection tube, and then replace the ordinary collection tube cap.

7.10 Transfer the droplets to an eight-tube strip, ensuring that the droplet surface does not exceed 100 μl. Cover the droplet surface with 100 μl of mineral oil. Cover the eight-tube strip with the cap and perform PCR according to the protocol in Table 11 (heated lid at 105°C), i.e., in-droplet PCR.

Table 11

(8) Demulsification and purification (based on 940-000792-00)

8.1 After the completion of the droplet PCR, transfer the droplet to a new low-adsorption 1.5 ml centrifuge tube, add 100 μl scATAC Breakage Reagent, and mix gently.

8.2 Remove the DNA Clean Beads in advance, equilibrate them at room temperature for at least 30 minutes, and shake them thoroughly before use.

8.3 Add 1.2 times the volume of the droplet (i.e. 1.2×, about 250 μl) of DNA Clean Beads to the droplet, vortex to mix thoroughly, and incubate at room temperature for 8 minutes.

8.4 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3-5 minutes until the liquid is completely clear and discard the supernatant.

8.5 Keep the centrifuge tube on the magnetic stand, add 500 μl of 80% ethanol to wash the magnetic beads and the tube wall, and discard the supernatant.

8.6 After washing the magnetic beads twice with ethanol, centrifuge the tube briefly and place it on a magnetic rack. Use a pipette to remove as much liquid as possible and dry the magnetic beads at room temperature.

8.7 After the magnetic beads are dried, add 50 μl of NF-H ₂ O, vortex to mix, and incubate at room temperature for 5 minutes.

8.8 Place the magnetic beads on a magnetic stand and transfer the supernatant to a new PCR tube to obtain cDNA, gDNA, and mtDNA.

(9) Isolation of cDNA, gDNA, and mtDNA

9.1 Thoroughly shake and mix MyOne ^™ Streptavidin C1 magnetic beads (i.e., streptavidin magnetic beads, cat. no. 65001, Invitrogen). Take out 10 μl of each sample and place it into a 200 μl PCR tube. Place it on a magnetic rack and let it stand until the liquid is clear. Discard the supernatant.

9.2 Add an appropriate amount of 1× Binding & Washing Buffer (prepare by diluting 2× Binding & Washing Buffer in Table 11 below with NF-H ₂ O) to tube 1, pipette to mix thoroughly to wash the magnetic beads, let stand until the liquid is clear, and discard the supernatant.

Table 11

9.3 After washing the magnetic beads three times, add 50μl of 2×Binding&washing Buffer to each sample to resuspend, add to the product in 8.8, vortex until completely mixed, and place on a rotary mixer to incubate for 30-60 minutes.

9.4 After incubation, place the PCR tube on a magnetic rack. After the supernatant is clarified, aspirate it and transfer it to a new PCR tube. This is the gDNA and mtDNA product to be purified. This step separates gDNA and mtDNA from cDNA.

(10) cDNA amplification and purification

10.1 Wash the magnetic beads in the tube twice with 1×Binding&washing Buffer.

10.2 Add appropriate amount of TE buffer to wash the magnetic beads. After the liquid is clear, discard the supernatant and add the PCR products listed in Table 12 below to the tube. Mix (primer sequence is the same as step 3.3).

Table 12

10.3 Perform PCR amplification. The reaction procedure is as shown in the table below to amplify the cDNA attached to the magnetic beads outside the droplets.

Table 13

10.4 Place the PCR product from step 10.3 on a magnetic rack and let it stand for about 5 minutes until the liquid becomes clear. Carefully pipette the supernatant into a new centrifuge tube.

10.5 Add 0.8X (80 μl) of DNA Clean Beads to the tube for purification, vortex to mix, centrifuge, and let stand at room temperature for 5 minutes;

10.6 Wash the magnetic beads according to steps 8.4 to 8.6;

10.7 After the magnetic beads have dried, add 20 μl of TE buffer to resuspend the beads, vortex to mix, and incubate at room temperature for 5 minutes to elute the cDNA. Transfer the supernatant to a new PCR tube to obtain the purified cDNA product.

10.8 Use Qubit to measure the concentration of the purified cDNA product.

(11) Droplet amplification and purification of gDNA and mtDNA

11.1 Add 50 μl of DNA Clean Beads to the supernatant separated in 9.4, vortex until completely mixed, and incubate at room temperature for 10 min.

11.2 After a brief centrifugation, place the PCR tube on a magnetic rack and transfer the supernatant to a new PCR tube;

11.3 Add 50 μl of DNA Clean Beads to the PCR tube, vortex to mix, and incubate at room temperature for 5 min.

11.4 After a brief centrifugation, discard the supernatant and wash the magnetic beads twice with 80% ethanol and allow the beads to dry.

11.5 Add 47 μl of NF-H2O, vortex thoroughly to mix, and incubate at room temperature for 5 min;

11.6 Place the PCR tube on a magnetic rack, transfer the supernatant to a new PCR tube, and add the reaction system: ATAC Enzyme III 50μl, scATAC Barcode Primer (940-000910-00) 4ul.

Perform PCR amplification. The reaction procedure is as follows:

Table 14

11.7 After PCR, add 1× (equal volume to the PCR product, 100 μl) DNA Clean Beads to purify the product. Follow the same steps as 11.2-11.5.

11.8 Place the PCR tube on a magnetic rack and transfer the supernatant to a new PCR tube. This is the purified mtDNA and gDNA.

11.9 Measure the product concentration using Qubit.

(12) High-throughput sequencing

12.1 cDNA Shedding

For the cDNA product, 100-150 ng was taken out and sheared according to the instruction manual of MGI's C series single-cell RNA library preparation kit (940-000510-00) to obtain cDNA fragments of about 500 bp for second-generation sequencing.

12.2 Library Construction

Following the instructions for the MGI C-Series Single-Cell RNA Library Preparation Kit (940-000510-00), the sheared cDNA, gDNA, and mtDNA products were denatured, single-stranded circularized, and then digested with enzymes. The denaturation process used the splint oligo primer sequence 5′-GCCATGTCGTTCTGTGAGCCAAGG-3′ (SEQ ID NO: 5). The digestion products were purified using silica-based magnetic beads.

12.3 Library Quality Inspection and Sequencing

The purified product was tested for concentration using qubit. A concentration > 0.5 ng/μl qualified the product and allowed for sequencing.

(13) Sequencing results analysis

13.1 Targeting transcriptome libraries and chromatin open regions

This example used an optimized formaldehyde concentration (0.1%) to fix the cells and a milder lysis condition, 0.1% NP40. Data analysis of the library constructed using this method revealed that in the transcriptome library, two replicates (samples PBMC_HB5 and PBMC_HB6) showed a good distribution trend in terms of UMI and gene detection. And the distribution trend difference between the two samples is extremely small (Figure 4); similarly, in the ATAC library, the high-quality library constructed by the method proposed in the embodiment of the present application can also be used for the detection of open chromatin regions, and effectively detect epigenetic information such as TSS enrichment, and the difference between samples is extremely small (Figure 5), indicating that the library construction method proposed in the embodiment of the present application can be effectively used to process samples that are more difficult to process, such as PBMC, and the constructed library is of high quality and highly stable among multiple samples, and can be used to stably construct high-quality libraries.

Figures 6 and 8 are transcriptome cluster diagrams for sample PBMC_HB5 and ATAC cluster diagrams for PBMC_HB6, respectively. As shown in Figures 6 and 8, the sequencing results of the libraries constructed using the methods of the present application examples can be effectively used to isolate cell subpopulations, and each cell subpopulation is clearly classified. As shown in Figures 7 and 9, the analysis results show that there is little batch variation between samples, indicating that the methods proposed in the present application examples can be effectively used for library construction of samples that are more difficult to process, such as PBMCs, and that the constructed libraries are of high quality, strong stability, and high reproducibility.

13.2 Targeting the mitochondrial genome

Table 15 shows the ATAC sequencing results of this example using a mild lysis solution containing only 0.1% NP40 as a lysis component (experimental group) and a conventional lysis solution containing 0.1% Tween-20, 0.01% digitonin and 0.1% NP40 (i.e., the control group, the rest of the experimental steps were the same as the experimental group, and both followed the above-mentioned experimental protocol in this example).

Table 15

As can be seen from Table 15, the mitochondrial proportion of the experimental group was significantly higher than that of the control group (15.29% vs 1.26%), indicating that compared with the cell lysis conditions used in the conventional scATAC test process used in the control group, the method proposed in the embodiment of the present application uses mild cell lysis conditions to treat cells through optimized cell lysis components. The constructed library can effectively retain mitochondrial information, which is beneficial to the joint analysis of the mitochondrial genome and its epigenomics and transcriptomics.

In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims (22)

A method for constructing a sequencing library for mtscCAT-seq based on droplet microfluidics, comprising: Cells were fixed and permeabilized using fixatives and lysis agents, respectively; Treating the fixed and permeabilized cells with a transposase, wherein the transposase is embedded with a first specific tag sequence to obtain gDNA and mtDNA connected to the first specific tag sequence, wherein the gDNA is derived from an open chromatin region; treating the cell with a transcriptome capture sequence, wherein the transcriptome capture sequence comprises a second specific tag sequence, so as to obtain cDNA connected to the second specific tag sequence by reverse transcription; generating droplets based on droplet microfluidics to enclose the cells and first microbeads in the droplets, wherein the gDNA, the mtDNA, and the cDNA are captured by the first microbeads; and Sequencing libraries for mtscCAT-seq were obtained based on captured gDNA, mtDNA, and cDNA. The method according to claim 1, wherein the fixative is formaldehyde, optionally with a final concentration of 0.1%-0.5%, preferably with a final concentration of 0.1%. The method according to claim 1 or 2, wherein the cleavage agent is ethylphenyl polyethylene glycol (NP40) and/or an NP40 substitute, and the NP40 substitute is polyoxyethylene octylphenol ether, branched (Octylphenoxy poly(ethyleneoxy)ethanol, branched), optionally with a final concentration of 0.1% NP40 and/or the NP40 substitute. The method according to claim 1, wherein the surface of the first microbead is connected to a microbead tag sequence, and the microbead tag sequence binds to the first specific tag sequence of the gDNA and the mtDNA and the second specific tag sequence of the cDNA, respectively, to capture the gDNA, mtDNA and cDNA, Wherein, the microbead tag sequence is microbead specific. The method according to claim 4, wherein after the gDNA, mtDNA, and cDNA are captured by the first microbeads, the method further comprises: Based on the binding of the microbead tag sequence of the first microbead with the gDNA, mtDNA and cDNA, the gDNA, mtDNA and cDNA are amplified in the droplet to obtain amplification products, wherein the amplification products include gDNA and mtDNA simultaneously connected to the microbead tag sequence and the first specific tag sequence, and cDNA simultaneously connected to the microbead tag sequence and the second specific tag sequence. The method according to claim 5, wherein after performing the in-droplet amplification, the method further comprises: disrupting the droplets to release the amplification products, Optionally, the method further comprises purifying the amplified product, and optionally, using second microbeads to purify the amplified product, wherein the second microbeads are optionally silica-based microbeads. The method according to claim 5 or 6, further comprising: The cDNA is separated from the gDNA and mtDNA using a third microbead, wherein the surface of the third microbead is connected to an attachment molecule, the cDNA contains an attachment molecule affinity sequence, and the attachment molecule specifically binds to the attachment molecule affinity sequence to separate the cDNA from the gDNA and mtDNA, Optionally, the attachment molecule is streptavidin, and the attachment molecule affinity sequence is a biotin sequence, Optionally, the biotin sequence is contained within the second specific tag sequence of the cDNA. The method according to any one of claims 1 to 7, further comprising: performing droplet-external amplification on the gDNA, mtDNA, and cDNA. The method according to any one of claims 1 to 8, wherein obtaining a sequencing library for mtscCAT-seq based on the captured gDNA, mtDNA, and cDNA specifically comprises: The captured gDNA, mtDNA and cDNA are subjected to library construction to obtain the sequencing library for mtscCAT-seq, Optionally, the method further includes: The cDNA is sheared, and a sequencing library is constructed based on the sheared cDNA. The method according to any one of claims 1 to 9, wherein the transposase is Tn5 transposase. The method according to any one of claims 1 to 10, wherein the first specific tag sequence comprises a first specific tag sequence a and a first specific tag sequence b, Optionally, the first specific tag sequence a is shown as SEQ ID NO: 1, and the first specific tag sequence b is shown as SEQ ID NO: 2. The method according to any one of claims 1 to 11, wherein the transcriptome capture sequence comprises polythymidine nucleotides (poly T) for capturing mRNA in the cell, Optionally, the transcriptome capture sequence is shown as SEQ ID NO: 3. The method of any one of claims 1 to 12, wherein the second specific tag sequence comprises a unique molecular identifier (UMI). The method according to any one of claims 1 to 13, wherein the microbead tag sequence comprises sequences i and ii, wherein the sequence i binds to all or part of the first specific tag sequence and the second specific tag sequence to capture the gDNA, mtDNA and cDNA, Optionally, the sequence ii comprises a microbead-specific tag sequence specific to a microbead, Optionally, the sequence ii further comprises a linker sequence. The method according to any one of claims 1 to 14, wherein obtaining a sequencing library for mtscCAT-seq based on the captured gDNA, mtDNA, and cDNA further comprises: Based on the captured gDNA, mtDNA, and cDNA, DNA nanoballs were prepared by rolling circle amplification to obtain sequencing libraries for mtscCAT-seq. The method according to any one of claims 1 to 15, wherein the cell is a mammalian cell, preferably a human cell, and the mammalian cell is derived from one or more cells in body fluids, body excretions, body secretions, lymphatic tissue, tonsils, bone marrow, muscle, liver, spleen, kidney, lung, heart, brain, intestine, stomach, pancreas, thymus, bladder and skin, Optionally, the cells are one or more cells selected from the group consisting of PBMCs, tonsil cells, lymphocytes, bone marrow cells, spleen cells and thymocytes. A droplet microfluidics-based mtscCAT-seq method comprising: Constructing a sequencing library according to the method for constructing a sequencing library for mtscCAT-seq based on droplet microfluidics according to any one of claims 1 to 16; and The library is sequenced, Optionally, the sequencing is second-generation sequencing and/or third-generation sequencing. Optionally, the sequencing is based on DNA nanoballs. A single-cell multi-omics analysis method based on mtscCAT-seq, including: Performing mtscCAT-seq according to the droplet microfluidics-based mtscCAT-seq method of claim 17 to obtain mtscCAT-seq data; and The mtscCAT-seq data were analyzed, and single bead tag sequences were obtained based on the mtscCAT-seq data. Comprehensive multi-omics information on cellular chromatin accessibility, transcriptome, and mitochondrial mutations. The method according to claim 18, wherein the comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome, and mitochondrial mutations is obtained based on the microbead tag sequences in the mtscCAT-seq data, comprising: distinguishing gDNA, mtDNA, and cDNA originating from the same cell based on the bead tag sequence of the first bead, wherein gDNA, mtDNA, and cDNA containing the same bead tag sequence are determined to originate from the same droplet and the same cell, and gDNA, mtDNA, and cDNA containing different bead tag sequences are determined to originate from different droplets and different cells; and The gDNA, mtDNA and cDNA derived from the same cell are analyzed to obtain comprehensive multi-omics information of chromatin accessibility, transcriptome and mitochondrial mutations of the single cell. A high-throughput single-cell multi-omics analysis method based on mtscCAT-seq, including: The method for constructing a sequencing library for mtscCAT-seq based on droplet microfluidics according to any one of claims 1 to 16, wherein m cells and n first microbeads are enclosed in a single droplet, wherein m≤1 and n≥1, preferably 1≤n≤4; sequencing the library to obtain mtscCAT-seq data; and The mtscCAT-seq data are analyzed, and comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome, and mitochondrial mutations is obtained based on the first specific tag sequence, the second specific tag sequence, and the microbead tag sequence in the mtscCAT-seq data. The method according to claim 20, wherein the comprehensive multi-omics information of single-cell chromatin accessibility, transcriptome, and mitochondrial mutations is obtained based on the first specific tag sequence, the second specific tag sequence, and the microbead tag sequence in the mtscCAT-seq data, comprising: Distinguishing gDNA, mtDNA, and cDNA originating from the same droplet based on the microbead tag sequence; Determining, based on the first specific tag sequence and the second specific tag sequence, gDNA, mtDNA, and cDNA in the same droplet originating from the same cell; and The gDNA, mtDNA, and cDNA derived from the same cell are analyzed to obtain comprehensive multi-omics information on chromatin accessibility, transcriptome, and mitochondrial mutations in the single cell. A single-cell multi-omics combined analysis method, comprising: The single-cell multi-omics analysis method based on mtscCAT-seq according to claim 18 or 19 or the high-throughput single-cell multi-omics analysis method based on mtscCAT-seq according to claim 20 or 21 for transcriptomics and/or Epigenomic and/or mitochondrial mutation analysis; and Combined analysis based on genomics, proteomics and/or metabolomics, Optionally, the epigenomics includes chromatin accessibility and one or more of the following: histone modification, DNA methylation, RNA methylation and non-coding RNA.