A high-throughput single-cell sequencing method based on a dual-pass microporous chip

Abstract

This invention discloses a method for constructing and sequencing single-cell multi-omics libraries based on a dual-permeable microarray design, comprising the following steps: S1, performing an in-situ nucleic acid molecular labeling reaction on a single-cell or mononuclear suspension of the tissue to be tested in a multi-well plate to tag it with a known base sequence; S2, mixing the reacted single-cell or mononuclear suspension with microbeads carrying the known base sequence and loading it onto a dual-permeable microarray; after the reaction is complete, sealing the chip with sealing oil; S3, performing nucleic acid molecule polymerization and amplification within the chip, and then collecting the liquid within the chip to construct a single-cell sequencing library; S4, sequencing the single-cell library and processing the data to obtain single-cell multi-omics library data. This invention enables high-throughput analysis of multi-omics genetic information, including transcriptomics and chromatin availability, at the single-cell level at a low cost and with rapid convenience.

Description

Technical Field

[0001] This invention belongs to the field of single-cell gene sequencing analysis technology, and specifically relates to a high-throughput single-cell sequencing method based on a dual-permeable microporous chip. Background Technology

[0002] Cellular heterogeneity has long been a challenge for developmental biologists and oncology researchers because population-level sequencing only provides average gene expression across different cell types, thus masking cell type-specific expression profiles. The rapidly developing single-cell sequencing technology effectively addresses this problem. It primarily relies on microfluidic or micropore systems to lock individual cells into a single reaction space, followed by the construction of next-generation sequencing libraries, allowing for the production of thousands to tens of thousands of cells in a single experiment. For example, Drop-seq (Macosko, EZ, et al., Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell, 2015. 161(5): p. 1202-1214.) and inDrop-seq (Klein, Allon M., et al., Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic StemCells. Cell, 2015. 161(5): p. 1187-1201.) platforms, which are mainly based on microfluidics, and Microwell-seq (Han, X., et al., Mapping the Mouse Cell Atlas by Microwell-Seq. Cell, 2018. 173(5): p. 1307.) and Seq-well (Gierahn, TM, et al., Seq-Well: Portable, Low-cost RNAsequencing of Single Cells) platforms, which are mainly based on microplates. Cells at high throughput. Nat Methods, 2017.14(4):p.395-398.) and the common commercial platform 10x Genomics Chromium Single Cell Gene Expression Solution. However, taking the 10x microfluidic platform as an example, when the cell loading exceeds the recommended loading amount, there will be two or more cells in one droplet at the same time, which makes it impossible to distinguish multiple cells in the same droplet in subsequent analysis; in order to control that there is only one cell in each droplet, the cell loading concentration must be strictly controlled, which will result in most droplets being empty, forming "empty load", which greatly reduces experimental throughput and cell utilization.

[0003] The above techniques are all based on sequencing live cells after isolation, digestion, or sorting. In the past year or two, another approach has also been adopted for single-cell sequencing: fixing the cells and then performing in situ transcript synthesis and sequencing. The principle is similar to in situ hybridization after tissue sectioning. However, in situ hybridization only uses fluorescent oligonucleotide probes to identify specific transcripts and cannot sequence all transcripts in a single cell. Sci-RNA-seq([1]Cao,J.,et al.,Comprehensive single-cell transcriptional profiling of a cellular multiorganism.Science,2017.357(6352):p.661-667.[2]Cao,J.,et al.,The single-cell transcriptional landscape of mammalian organogenesis.Nature, 2019.566(7745):p.496-502.) and SPLiT-seq (Rosenberg, AB, et al., Single-cell profiling of the developing mouse brain and spinal cord with split-pool (barcoding. Science, 2018, 360(6385): p.176-182.) They first fixed and permeabilized fresh cells, then evenly distributed the cells into multi-well plates, and used pore-specific reverse transcription primers to tag the cells in the first round. Subsequently, the cells were collected and mixed, and then evenly distributed into new multi-well plates with new tags. Finally, individual cells were distinguished through a combination of multiple rounds of molecular tags. However, since the subsequent tag introduction is based on intracellular connection reactions, there are problems such as low reaction efficiency and easy leakage of transcripts between cells. Therefore, the sensitivity and contamination rate of these platforms have considerable room for improvement. Paul et al. from the Center for Molecular Medicine Research at the Austrian Academy of Sciences (Datlinger, P., et al., Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing. Nature Methods, 2021.) were the first to apply the combinatorial indexing approach to a microfluidic platform. They used reverse transcription to attach a pre-tag containing 96 species to immobilized cells, and then combined this with microfluidic methods to increase the throughput of single-cell sequencing by 15 times.However, microfluidic-based sequencing platforms do not allow for parallel experiments, resulting in significant batch effects. They also suffer from drawbacks such as expensive equipment, difficulty in portability, and high sequencing costs.

[0004] Besides transcriptomics, chromatin transposase accessibility analysis (ATAC) has also gradually shifted from batch population sequencing to the single-cell level in recent years. Unlike transcriptomics, ATAC sequencing uses transposases to identify open regions of chromatin without nuclei, cleaving them to produce fragments of specific lengths. This determines the open state of the entire cellular genome, providing information on chromatin accessibility regions and revealing actively transcribed regions. The mainstream approaches to single-cell ATAC-seq are also divided into two types: one is to first isolate single cells using droplets or microwells, then perform transposase digestion to attach specific tags and sequencing adapters, obtaining an amplified library; the other is based on combinatorial indexing, which introduces the first round of tags through transposition reactions, followed by subsequent tags through ligation reactions or PCR amplification, ensuring a sufficient number of tag combinations to distinguish individual cell combinations. Studies have reported sci-ATAC-seq and sci3-ATAC-seq, providing single-cell ATAC studies with throughputs of tens of thousands and hundreds of thousands of cells, respectively. Similar to Sci-RNA-seq, sci-ATAC-seq is limited by the number of combinations, has low throughput, and a high rate of intercellular contamination. sci3-ATAC-seq, building upon the former, employs a method of two consecutive rounds of ligation after transposase digestion without tags, increasing the number of combinations. However, it sacrifices some sensitivity due to the efficiency issues of two rounds of ligation in vivo. Furthermore, all of the above-mentioned combinatorial labeling methods require ligases to perform oligonucleotide ligation labeling. The cost of ligases required for large-scale experiments is relatively high.

[0005] In existing technologies, microwell single-cell sequencing methods generally rely on gravity sedimentation to capture single cells and magnetic beads into microwells. However, the Poisson distribution characteristic of microwell gravity sedimentation limits cell detection efficiency. Some digital PCR systems utilize dual-channel microwell arrays based on capillary action and liquid absorption, avoiding the inefficiencies caused by gravity sedimentation and Poisson distribution. However, the thickness and materials of the dual-channel microwell arrays used in digital PCR are not suitable for direct application in single-cell omics methods. Furthermore, the uniform liquid capillary action results in multiple beads or cells within each microwell, making direct analysis of single-cell resolution omics data impossible. Therefore, developing a low-cost, high-throughput, semi-open system with high adaptability for single-cell multi-omics sequencing is crucial to meeting a wide range of analytical needs. Summary of the Invention

[0006] The main objective of this invention is to propose a high-throughput single-cell multi-omics sequencing method based on a dual-permeable microwell chip. This method can obtain specific omics information (such as full-length transcriptome, epigenome, proteome, etc.) of hundreds of thousands of single cells in a single batch. This method is applicable to fresh, frozen, or embedded tissues and is not limited by cell viability or state. This invention is based on a dual-permeable microwell array design and provides a decoding and reconstruction strategy for multiple microbeads and multiple cells existing in different microwells based on sequencing sequence similarity analysis, establishing a single-cell multi-omics sequencing method based on a dual-permeable microwell chip.

[0007] To achieve the above objectives, this invention proposes a high-throughput single-cell multi-omics sequencing method based on a dual-permeable microporous chip, comprising the following steps:

[0008] S1. In situ nucleic acid molecular labeling reaction is performed on single-cell or mononuclear suspensions of the tissue to be tested in a multi-well plate to tag them with a known base sequence;

[0009] S2. Mix the reacted single-cell or mononuclear suspension with microbeads containing known base sequences and load them onto a double-permeable microporous chip. After the reaction is complete, seal the chip with sealing oil.

[0010] S3. Nucleic acid molecules are polymerized and amplified within the chip, and then the liquid within the chip is collected to construct a single-cell sequencing library;

[0011] S4. Sequencing of single-cell libraries and data processing to obtain single-cell multi-omics library data. This invention enables rapid, convenient, and high-throughput analysis of multi-omics genetic information, including transcriptomics and chromatin availability, at the single-cell level at a low cost.

[0012] This invention provides a method for constructing a single-cell omics library based on a dual-permeable microporous chip. By introducing a first cell identity tag through in situ reaction of cells / nuclei, multiple microbeads and cells / nuclei are loaded onto the dual-permeable microporous chip, allowing them to be uniformly and rapidly drawn into the micropores through capillary action. The second cell identity tag on the microbeads is then incorporated into the nucleic acids of the cells / nuclei. Nucleic acid polymerization and amplification reactions are performed within the chip, enabling the reconstruction and analysis of multi-omics genetic information of single cells at the single-cell level. Attached Figure Description

[0013] To more clearly illustrate the embodiments of the present invention and the technical solutions in the prior art, the accompanying drawings involved in the description of the embodiments and the prior art will be briefly introduced below. The following drawings are only some embodiments of the invention. For those skilled in the art, without creative effort, they can repeat the solution of the present invention and obtain other drawings based on the data obtained by the method shown in the drawings.

[0014] Figure 1 This is a schematic diagram of the experimental process in one embodiment of the present invention.

[0015] Figure 2 This is a schematic diagram of the process for transcriptome experiments using single-cell nucleus suspension in one embodiment of the present invention.

[0016] Figure 3 This is a schematic diagram showing the distribution of microbeads and cell nuclei trapped in the micropores of a chip through capillary action in one embodiment of the present invention.

[0017] Figure 4 This is a contamination ratio diagram of a transcriptome data mixing experiment between human and mouse species cells in one embodiment of the present invention.

[0018] Figure 5 This is a diagram showing the distribution ratio of transcripts across the full length of a gene in transcriptome data according to one embodiment of the present invention.

[0019] Figure 6 This invention provides a correlation score for different microbead pairs after sequence similarity calculation, as well as a threshold for microbeads that need to be merged, in one embodiment of the invention.

[0020] Figure 7 This is a schematic diagram of the structure of an epigenomic sequencing library using a single-cell nucleus suspension in one embodiment of the present invention.

[0021] Figure 8 This is a contamination ratio diagram of a human and mouse species cell mixture experiment in epigenomic sequencing library data according to an embodiment of the present invention.

[0022] Figure 9 This is a type annotation of open fragments of the human (left) and mouse (right) genomes captured in epigenomic sequencing library data according to an embodiment of the present invention. Detailed Implementation

[0023] To more clearly illustrate the implementation objectives, technical solutions, and advantages of this invention, the technical solutions in the embodiments of this invention will be described clearly and completely below. Where specific conditions are not specified in the embodiments, they are generally performed according to conventional conditions or conditions recommended by the instrument / reagent manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. The term "and / or" in the entire description includes parallel solutions that are satisfied individually or simultaneously. The technical solutions of the various embodiments can be combined with each other, but only on the premise that they can be implemented by those skilled in the art. When the combination of solutions results in contradictions or cannot be implemented, it should be considered that the technical solution is not within the protection scope of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this invention.

[0024] The technical solution of this invention utilizes high-throughput single-cell capture based on a dual-permeable microporous chip to simultaneously obtain specific omics information (such as full-length transcriptome, epigenome, proteome, etc.) of hundreds of thousands of single cells. This method is applicable to fresh, frozen, or embedded tissues and is not limited by cell viability or state. This invention is based on a dual-permeable microporous array design and provides a decoding and reconstruction strategy for multiple microbeads and multiple cells existing in different micropores based on sequencing sequence similarity analysis, establishing a single-cell multi-omics sequencing method based on a dual-permeable microporous chip.

[0025] To achieve the above objectives, this invention proposes a high-throughput single-cell multi-omics sequencing method based on a dual-permeable microporous chip, such as... Figure 1 As shown, in this embodiment, the following steps are included:

[0026] S1. In situ nucleic acid molecular labeling reaction is performed on single-cell or mononuclear suspensions of the tissue to be tested in a multi-well plate to tag them with a known base sequence;

[0027] S2. Mix the reacted single-cell or mononuclear suspension with microbeads containing known base sequences and load them onto a double-permeable microporous chip. After the reaction is complete, seal the chip with sealing oil.

[0028] S3. Nucleic acid molecules are polymerized and amplified within the chip, and then the liquid within the chip is collected to construct a single-cell sequencing library;

[0029] S4. Sequencing of single-cell libraries and data processing to obtain single-cell multi-omics library data. This invention enables rapid, convenient, and high-throughput analysis of multi-omics genetic information, including transcriptomics and chromatin availability, at the single-cell level at a low cost.

[0030] Furthermore, in step S1, nucleic acid molecules or proteins with known base sequences are added to different wells of the 96-well plate, while nucleic acid molecules or proteins with the same known base sequence are added to the same well. This allows for in-situ mRNA reverse transcription / epigenomic fragmentation within the cell / nucleus while simultaneously attaching the first cell identification tag to the cell / nucleus, facilitating rapid and accurate identification of cells / nuclei from different samples during subsequent analysis.

[0031] Furthermore, step S1 further includes: resuspending and fixing the cell / nucleus suspension using any one of the following fixatives: aldehyde fixative (such as paraformaldehyde), alcohol fixative (such as ethanol), acid fixative, and cross-linking agent. This allows the nucleic acids / proteins within the cells / nucleus to cross-link and be fixed, enabling nucleic acid molecules to more effectively enter the cells / nucleus for reaction. Preferably, in chromatin accessibility sequencing, the cell nucleus is not fixed in any way, allowing transposases to more effectively enter the cell nucleus for reaction.

[0032] In some embodiments of the present invention, in step S1, the molecules that bind to mRNA in the cell / nucleus include single-stranded oligonucleotides with known base sequences, and the molecules that bind to DNA in the cell / nucleus include transposases with known base sequences.

[0033] Furthermore, in step S2, the micropore shape of the dual-through-micropore chip includes any one of circular, rectangular, or regular hexagonal shapes. The pore diameter is between 10µm and 1mm. Specifically, it can be between 20µm and 100µm, preferably between 40µm and 60µm. Different micropore shapes or sizes are selected according to the cell / nucleus and molecule size, as well as the need for adjustment or modification of the micropore spacing on the chip surface.

[0034] Furthermore, in step S2, the material of the dual-channel through-hole chip includes any one of glass fiber, polymer, and silicon wafer. Different materials can be selected for chip fabrication according to the dual-channel etching process requirements corresponding to the chip size and thickness.

[0035] Furthermore, in step S2, the material of the microbeads includes any one of polymer microbeads, magnetic microbeads, hydrogel microbeads, and biodegradable polymer microbeads. Depending on the requirements, nucleic acid analysis components are cross-linked and connected to the surface of the microbeads or polymerized inside the biodegradable microbeads. The diameter of the microbeads varies depending on the micropore size, specifically between 3µm and 100µm.

[0036] Furthermore, in step S2, the nucleic acid molecules with known base sequences connected to the surface of the microbeads include conditionally cleavable sites, wherein the conditionally cleavable sites include any one of the following: dU base modification, disulfide bond modification, photocleavable linker, and restriction endonuclease recognition sequence. Different cleavage modes can be selected as needed to facilitate the release of nucleic acid molecules into the micropores for reaction.

[0037] Furthermore, in step S2, the direct coupling of the nucleic acid molecule with the known base sequence to the microbeads includes any one of the following: condensation acylation reaction based on carboxyl amino groups, cross-linking reaction based on biotin and streptavidin, ester bond linkage, disulfide bond linkage, and dipole interaction. Different polymerization methods can be selected as needed to facilitate the release of nucleic acid molecules into the micropores for reaction.

[0038] In some embodiments of the present invention, in step S2, the surface of the microbeads contains carboxyl group modifications, the nucleic acid molecule with a known base sequence contains amino group modifications, and the conditionally cleavable site includes dU base modifications. The nucleic acid molecule with a known base sequence is attached to the surface of the magnetic beads via an acylation reaction, and the nucleic acid molecule is released into the microporous liquid environment for reaction by cleavage of the dU base.

[0039] In some embodiments of the present invention, in step S1, the targets of the in situ nucleic acid molecular labeling reaction in the cell / nucleus are mRNA and DNA. In step S2, nucleic acid molecules with known base sequences on the surface of the microbeads carrying oligo-dT can hybridize and bind to mRNA in the treated cells / nucleus; the fixed hybridization sequence carried by nucleic acid molecules with known base sequences on the surface of the microbeads can hybridize and bind to DNA in the treated cells / nucleus.

[0040] Furthermore, in step S2, the multi-segment extension of known base nucleic acid molecules on the surface of the microbeads includes split-pool segmented PCR hybridization extension amplification and split-pool segmented ligase ligation extension, which can be selected according to actual conditions.

[0041] In some embodiments of the present invention, in step S2, after the microbeads and cells are loaded onto the chip, the evaporation of liquid on the chip surface can be promoted by natural evaporation, vacuum evaporation, or other methods, making the reaction compartments between the micropores more independent and reducing molecular contamination. In the epigenomic sequencing method, the chip is heated at a constant temperature to release DNA from the cell nucleus, so as to facilitate further hybridization reactions with nucleic acid molecules on the microbeads.

[0042] Furthermore, in step S3, the amplification methods for intracellular / nuclear molecules include any one of isothermal polymerization, isothermal amplification, thermal cycling PCR exponential amplification, and thermal cycling PCR linear amplification. Different polymerization and extension methods can be selected as needed.

[0043] Furthermore, in step S4, preferably, during the data analysis process, a similarity expression score is first calculated by the similarity of the random sequence distribution in the cell identity tags carried by the microbeads and captured in the data. This determines which microbeads are located in the same microwell, and the genetic sequence information of all microbeads in the same microwell is merged. For cells / nuclei in the same microwell, the genetic sequence information merged by the microbeads is allocated and restored to individual cells / nuclei using the cell / nucleus identity tags in step S1, thus obtaining omics data at single-cell resolution.

[0044] It should be noted that, in some embodiments of the present invention, the known base sequence tag mentioned in step S1, for transcriptome sequencing library construction, uses a reagent containing random oligonucleotide sequences for reverse transcription, characterized by its ability to randomly hybridize and pair with any region of nucleic acid, followed by unbiased reverse transcription of the full-length nucleic acid region; the primer sequence for microbead ligation of the transcriptome sequencing library mentioned in step S3 includes four parts: a library adapter sequence, cell tag 2 formed by a combination of three segments of 96 fixed sequences, UMI, and Poly(dT) sequence. The library adapter sequence is used for subsequent sequencing; cell tag 2 and cell tag 1 are used together to identify different cells; the UMI is a sequence composed of random bases, and each DNA molecule contains a unique UMI, used to distinguish different DNA molecules during mixed sequencing; the Poly(dT) sequence is used to capture cDNA molecules containing polyA tails.

[0045] It should be noted that, in some embodiments of the present invention, the known base sequence tag mentioned in step S1, for epigenomic sequencing library construction, uses a transposase adapter containing a cell identity tag sequence to break open chromatin regions; the primer sequence for bead linking in step S3 for epigenomic sequencing library includes four parts: a library adapter sequence, a cell tag 2 formed by combining three segments of 96 fixed sequences, a UMI, and a transposase adapter sequence. The library adapter sequence is used for subsequent sequencing; cell tag 2 and cell tag 1 are used together to identify different cells; the UMI is a sequence composed of random bases, and each DNA molecule contains a unique UMI to distinguish different DNA molecules during mixed sequencing; the transposase adapter sequence is used to capture the DNA sequence broken by the transposase.

[0046] This invention also proposes a sequencing and data processing method, comprising the following steps:

[0047] Using the aforementioned cell / nucleus in situ labeled nucleic acid molecules, a single-cell library was constructed using a double-permeable microporous chip and microbeads to obtain transcriptome and epigenome libraries;

[0048] The obtained transcriptome and epigenome libraries were sequenced to obtain single-cell transcriptome data and chromatin accessibility data;

[0049] Based on the correspondence between the positional information and genetic information of in situ labeled nucleic acid molecules and nucleic acid molecules with known base sequences on microbeads, the genetic information of different samples in the multiple samples to be tested can be obtained.

[0050] Specifically, when performing high-throughput sequencing, the obtained transcriptome and epigenome libraries are sequenced separately or in combination. The sequencer can be an Illumina platform, a BGI platform, or other sequencing platforms that are compatible with the sequencing adapters. Furthermore, those skilled in the art can modify the oligonucleotide primer sequence information before the cell identity tag according to the requirements of different sequencing platforms to make it suitable for different sequencers. These primer sequence modifications do not affect the scope of protection of this invention.

[0051] Specifically, when performing high-throughput sequencing data analysis, a valid cell dataset is first obtained based on the cell identity tags in the in-situ labeled nucleic acid molecules described in step S1 and the nucleic acid molecules with known base sequences contained in the microbeads described in step S1. The genetic information of single-cell data is then reconstructed through nucleic acid sequence similarity calculations in the data described in step S4, and the RNA expression and DNA copy number of the corresponding cells are detected based on the molecular tag sequences.

[0052] The library construction and sequencing method provided by this invention has all the technical solutions of the single-cell multi-omics construction method based on dual-permeable microporous chip, and therefore has all the beneficial effects of the single-cell multi-omics construction method based on dual-permeable microporous chip, which will not be elaborated further in this invention.

[0053] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain part of the content of the present invention and are not intended to limit the present invention.

[0054] Example 1: Preparation of molecularly labeled microbeads:

[0055] Molecular marker sequences are attached to the surface of the microbeads. This step employs a "split-pool" strategy of "dispersion-merging-dispersion" to prepare microbeads with multiple nucleic acid markers. For specific preparation methods, please refer to patent CN106498040B. The steps are as follows:

[0056] Nucleic acid molecules with known base sequences were designed and divided into three segments. PCR hybridization adapter sequences were placed between adjacent segments. The first segment, starting at 5', included amino modifications, a fixed library adapter sequence, and some cell identity tag sequences. For the transcriptome, the 3' segment of the microbeads contained some cell tag sequences, molecular tag sequences, and poly-T tails; for the epigenome, the 3' segment of the microbeads contained some cell tag sequences, molecular tag sequences, and hybridization sequences. Ninety-six cell tag sequences were designed for each segment, each placed independently. An amino group was used to replace a hydroxyl group at the C6 position of the nucleotide at the 5' end of the first segment (5NH2C6). Equal amounts of carboxyl-modified microbeads were coupled to each of the 96 first segment sequences. The resulting 96 modified magnetic beads were collected, mixed thoroughly, and then divided into 96 equal portions. Each portion was then mixed with 96 second segment sequences and subjected to PCR sequence extension. The mixture was then divided into 96 equal portions and mixed with 96 third segment sequences and subjected to PCR sequence extension. Finally, denaturation and melting were performed to obtain magnetic beads with 884,736 single-stranded oligonucleotide modifications.

[0057] Example 2: Species Mixed Cell Experiment

[0058] Sample preparation

[0059] according to Figure 1 The methodology involved constructing single-cell transcriptome sequencing libraries using human 293T and mouse 3T3 cell lines. The library structures are as follows: Figure 2 As shown. The two cell lines were washed once with 1×PBS solution, and then resuspended in 1 ml of lysis buffer (RSBT wash buffer containing 0.1% IGEPAL CA-630). They were placed on ice for lysis for 3 min, and the lysis reaction was terminated by adding 5 mL of RSBT wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% RNase inhibitor).

[0060] Reverse transcription

[0061] Cell nuclei were fixed using 4% PFA and resuspended in PBST. A reverse transcription reagent containing reverse transcriptase (Thermo Fisher Scientific, USA), reverse transcription reaction buffer, dNTPs (Sole ProBio, Beijing), random reverse transcription primers (synthesized by Shanghai Sangon Biotech Co., Ltd.), RNase inhibitor (Nanjing Novizan Biotechnology Co., Ltd.), 10% Triton X-10 (Shanghai Sangon Biotech Co., Ltd.), and 50% PEG8000 (Shanghai Sangon Biotech Co., Ltd.) was added, mixed thoroughly, and then aliquoted into 96-well plates for reverse transcription. After the reverse transcription reaction, 10 μl of 40 mM EDTA was added, and the reaction was incubated at 37°C for 15 minutes to terminate the reaction. The cells were then washed three times with 3×SSC and once with PBS.

[0062] external incision

[0063] After reverse transcription, the sample was added with exonuclease reagent containing EXO I exonuclease (purchased from NEB, USA), exonuclease reaction buffer, and RNase inhibitor (purchased from Nanjing Novizan Biotechnology Co., Ltd.), and incubated at 37°C for 30 minutes to remove excess random primers. Then it was washed three times with 3×SSC and once with PBS.

[0064] Add capture connector

[0065] The excision samples were added to a reaction system containing terminal transferase (purchased from NEB Corporation, USA), terminal transferase reaction buffer, RNase inhibitor (purchased from Nanjing Novizan Biotechnology Co., Ltd.), and dATP (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and incubated at 37°C for 30 minutes. After the reaction, the samples were washed three times with PBST.

[0066] Amplification reaction

[0067] 5 μl of cell nuclei (approximately 4 million / mL) were resuspended in approximately 30,000 molecularly labeled magnetic beads, followed by the addition of 1 μl of P7 adapter and 5 μl of 2× high-fidelity polymerase, and mixed thoroughly. This mixture was then loaded onto a custom-made hexagonal dual-channel chip (60 μm diagonal, 15 μm well spacing, 800 μm thickness) to ensure the overall distribution of magnetic beads and cell nuclei capture was as follows: Figure 3 As shown, the cell nuclei and microbead drop-off rate within the chip is greater than 70%. Subsequently, an amplification system containing isothermal polymerase, RNase H, USER enzyme, and high-fidelity polymerase was uniformly added to the reverse side of the first sample loading. The chip was then placed vertically in a centrifuge tube, and sealing oil was added to seal the microwells to create individual reaction spaces. The tube was then placed in a PCR thermal cycler for amplification.

[0068] Collect the liquid and purify it.

[0069] After the reaction was complete, the liquid and molecularly labeled magnetic beads in the chip were collected thoroughly by multiple centrifugations, placed on a magnetic rack, and the supernatant was transferred to a new reaction tube. The total volume (A) was measured with a pipette, and then 1.5A of DNA CleanBeads purification magnetic beads (purchased from Nanjing Novizan Biotechnology Co., Ltd.) was added for purification to obtain a cDNA solution.

[0070] Sequencing library amplification

[0071] The obtained cDNA was added to an amplification system containing tagged primers P5, P7 and high-fidelity polymerase for amplification to obtain a sequencing library with an index. The sequencing library was then purified using DNA Clean Beads, and the library concentration was determined using Qubit 3.0 fluorescent reagent. The library was stored at -20°C.

[0072] Data preprocessing

[0073] The sequencing library was sequenced using the BGIDNBSEQ-T7 sequencing platform in PE150 mode. The returned raw FASTQ data was extracted and screened based on cell tag sequences. Gene expression profiles were obtained by comparing Read2 data to human and mouse reference genomes. Further analysis was conducted on the number of transcripts detected in individual cells from different species and the presence of cross-species transcripts within individual cells. The results are as follows: Figure 4 As shown, the average number of transcripts for gene expression in both cell types is close to 3000, and the cross-contamination rate of transcriptomes between species is approximately 1%, indicating that this method has a low cell cross-contamination rate and high sensitivity in detecting transcripts. The overall captured transcripts are evenly distributed across the 5' to 3' ends of the entire gene, as shown... Figure 5 As shown, this indicates that the transcripts captured by the random primers are transcripts from the full length of the gene.

[0074] Multi-bead multi-cell decoding computation

[0075] Cell identity tag combinations from the gene expression matrix are filtered to remove low-quality cells (less than 500 molecular tags). Based on cell identity tags extracted from sequencing data by location, all microbeads are paired and arranged in a traversal manner. For each bead pair, a traversal pairing calculation is performed, calculating the similarity expression score of the captured sequence. In transcriptome sequencing, the sequences included in the calculation are random primer sequences; in chromatin accessibility sequencing, the calculated sequences are the captured genetic information. The microbeads are then sorted according to their similarity expression scores. Figure 6 Based on the actual number of pores contained in the microbeads, highly sequence-similar microbeads are paired and merged, and then genetic information is assigned to different cells based on cell identity tags.

[0076] Example 3: Species Mixed Cell Experiment

[0077] Sample and reagent preparation

[0078] according to Figure 1 Methodology: Using human 293T cell line and mouse 3T3 cell line, single-cell chromatin accessibility epigenome sequencing libraries were constructed. The library structure is as follows: Figure 7 As shown. Primers containing transposase-embedded recognition sequences and cell identification tag sequences (384 combinations) were placed in a PCR instrument at 95°C for 2 minutes and then cooled to 25°C at a rate of 0.1°C / second. The primers were diluted with enzyme-free water to prepare working solutions and aliquoted into multiple 96-well plates. A mixture containing Tn5 transposase, coupling buffer, and dilution buffer was added to the annealed primer working solution, thoroughly mixed, and incubated at 30°C for 1 hour. The incubated enzyme was then stored at -20°C.

[0079] Transposable enzyme digestion reaction

[0080] The mixed human and mouse cell nuclei were evenly distributed into 96-well plates. Then, an enzyme digestion system containing an embedded oligonucleotide cell-tagged transposase, 2× digestion buffer, 1% digitalis saponin, 10% Tween-20, and 1×PBS was added to each well. The mixture was thoroughly mixed and incubated at 55°C for 30 minutes. After the reaction was complete, the 96-well plates were removed and placed on ice for 5 minutes to terminate the digestion. Cells were collected from the 96-well plates using a multipipeline, centrifuged at 500g / 5min, and then washed twice with RSBT buffer.

[0081] Release enzyme fragments

[0082] Resuspend 5 μl of cell nuclei (approximately 4 million / mL) in approximately 30,000 molecularly labeled magnetic beads, then add 7 μl of 50 mM EDTA and 5 μl of 2× high-fidelity polymerase, and mix well. The mixture is then loaded onto a custom-made hexagonal double-sided microarray (60 μm diagonal, 15 μm well spacing, 800 μm thickness), ensuring a cell nucleus and microbead placement rate of over 70%. The tube is then capped and centrifuged at 50°C for 30 min to release the enzyme-digested genomic fragments. The resulting fragments are then amplified using a PCR thermal cycler.

[0083] Fragment release and amplification reaction

[0084] Take 30,000-40,000 molecularly labeled magnetic beads, wash twice with 1×PBS, and place on ice for later use. Adjust the cell density after enzyme digestion to 5 million / mL, and resuspend the molecularly labeled magnetic beads in 4 μl of cells. Mix 14 μl of the prepared mixture by pipetting, and quickly and evenly load it onto the chip using a pipette tip. Gently scrape the surface with the pipette tip until the liquid is completely absorbed. Gently remove the liquid from the chip surface using a vacuum pump to create a concave liquid surface in the microwells. Tighten the cap, and then incubate the centrifuge tube at 50°C for 30 min to release the enzyme-digested genomic fragments. Remove the chip, and evenly add amplification reagent to the reverse side of the first loading area, gently scraping the surface with the pipette tip until the liquid is completely absorbed. Seal the microwells with sealing oil to create individual reaction spaces, and then place the chip in a PCR thermal cycler for template amplification.

[0085] Collect the liquid and purify it.

[0086] After the reaction was complete, the liquid and molecularly labeled magnetic beads in the chip were collected thoroughly by multiple centrifugations, placed on a magnetic rack, and the supernatant was transferred to a new reaction tube. The total volume (A) was measured with a pipette, and then 1.5A of DNA CleanBeads purification magnetic beads (purchased from Nanjing Novizan Biotechnology Co., Ltd.) was added for purification to obtain a cDNA solution.

[0087] Sequencing library amplification

[0088] The obtained cDNA was added to an amplification system containing tagged primers P5, P7 and high-fidelity polymerase for amplification to obtain a sequencing library with an index. The sequencing library was then purified using DNA Clean Beads, and the library concentration was determined using Qubit 3.0 fluorescent reagent. The library was stored at -20°C.

[0089] Data preprocessing

[0090] Sequencing libraries were used on the BGIDNB-SEQ T7 sequencing platform in PE150 sequencing mode. Data analysis was performed as described in Example 2. The cross-contamination rate of species DNA alignment reads within single cells was analyzed, and the results are as follows: Figure 8 As shown, the cross-contamination rate of genome reads between species is approximately 0.7%, indicating that the cross-contamination rate among single-cell species for detecting the degree of chromatin openness in the genome using this method is extremely low. The number of single-cell reads specifically captured is close to 200,000, demonstrating very high specificity and sensitivity. Annotations were performed on the captured open region fragments in humans and mice, such as... Figure 9 As shown, a typical enrichment rate of 15% is within the promoter range, indicating that the captured open region fragments are correct.

[0091] In summary, this invention provides a high-throughput single-cell sequencing method based on a dual-permeable microporous chip. It utilizes in-situ cell / nucleus labeling and the capillary action of the dual-permeable microporous chip to rapidly and efficiently capture and isolate a large number of single cells. The genetic information of the single cells is amplified in one step within the chip, and then a sequencing library is constructed, thereby enabling high-throughput detection of multi-omics genetic information at the single-cell level.

[0092] The above are merely preferred embodiments of the present invention and do not limit the patent scope of 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 patent protection scope of the present invention.

Claims

1. A method for single-cell library construction sequencing based on a double-pass penetration type micro-well chip, the method comprising the following steps: S1.Preparing a single-cell or single-nucleus suspension from a tissue, uniformly distributing the suspension into a multi-well plate, and in situ labeling molecules with known base sequences in the cells or cell nuclei as part of a tag for labeling cell identity; S2.Mixing the reacted single-cell or single-nucleus suspension with microbeads with known base sequences as another part of the tag for labeling cell identity, and loading the mixture onto a double-pass penetration type micro-well chip, so that the cells / nuclei and the microbeads are quickly and uniformly absorbed into the micro-wells by capillary action, and one micro-well can accommodate multiple microbeads and cells / nuclei, and sealing the chip with sealing oil after the loading reaction is completed; the double-pass penetration type micro-well chip has an array of micro-wells, each of which is a double-pass hole penetrating the upper and lower surfaces of the double-pass penetration type micro-well chip, and the liquid absorption on the surface of the chip is based on rapid and uniform capillary action; S3.Performing nucleic acid molecule polymerization and amplification in a test tube or chip carrier containing the chip, and then collecting the liquid in the chip to obtain a pre-amplification library for single-cell sequencing; S4.Constructing a single-cell sequencing library and performing sequencing, and obtaining single-cell multi-omics library data through data processing.

2. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, The double-pass penetration type micro-well chip can be adapted to a 200 microliter eight-tube PCR tube, a 1.5 milliliter centrifuge tube; and / or The shape of the micro-well of the double-pass penetration type micro-well chip includes any one of a circle, a square, and a regular hexagon; And / or The double-pass penetration type micro-well chip can also be scaled up to different sizes and shapes, such as a circle, a trapezoid, and a rectangle, and a corresponding chip carrier is used to perform large-scale ultra-high-throughput single-cell / single-nucleus analysis; and / or The microbead material medium loaded with the double-pass penetration type micro-well chip includes any one of a polymer microbead, a magnetic microbead, a hydrogel microbead, and a degradable polymer microbead; and / or The material of the double-pass penetration type micro-well chip includes any one of glass fiber, a high polymer, and a silicon wafer; and / or The outer surface of the micro-well of the chip is subjected to hydrophobic treatment, and the inner wall of the micro-well is subjected to hydrophilic treatment.

3. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, In step S1: An aldehyde fixing solution, an alcohol fixing solution, and an acid fixing solution are used to fix the cells or cell nuclei; and / or In chromatin accessibility sequencing, the cell nuclei are not subjected to any fixing treatment.

4. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, In step S1: Different wells of the multi-well plate can be added with cells / nuclei from different samples to realize mixed sample labeling in one experiment; The in situ labeling object in the cells / nuclei is mRNA / DNA, and the molecules with known base sequences include nucleic acids; Or The in situ labeling object in the cells / nuclei is protein, and the molecules with known base sequences include nucleic acids modified on antibodies.

5. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, In step S2: The nucleic acid molecules with known base sequences contained in the microbeads include conditional cleavable sites, wherein the conditional cleavable sites include any one of a dU base modification, a disulfide bond modification, a photocleavable linker, and a restriction enzyme recognition sequence; and The nucleic acid molecules of the known base sequence are directly coupled to the microbeads in any one of the following manners: condensation acylation reaction based on carboxyl amino, cross-linking reaction based on biotin and streptavidin, ester bond connection, disulfide bond connection and dipole interaction.

6. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, In step S2: The method for multi-stage extension of the nucleic acid molecules of the known base sequence to the nucleic acid molecules already coupled on the microbeads includes any one of the following: multi-step PCR amplification and multi-step ligase ligation.

7. The double-pass penetration type microwell chip-based single-cell library construction sequencing method of claim 1, wherein, In step S2: The micro-wells can simultaneously accommodate multiple microbeads and multiple cells / nuclei, and the cells / nuclei and the microbeads are quickly adsorbed into the micro-wells by capillary action with the liquid on the surface of the chip, rather than gravity sedimentation and Poisson distribution.

8. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, In step S3: In the chip sealed by the sealing oil, the intracellular nucleic acid molecules are polymerized and pre-amplified by heat transfer, and the pre-amplification method includes any one of exponential PCR amplification and linear PCR amplification.

9. The double-pass penetration type microwell chip-based single cell library construction sequencing method of claim 1, wherein, In step S4: For the case where multiple microbeads, multiple cells / nuclei exist in the same micro-well, the microbeads and the cells / nuclei located in the same micro-well are determined by expressing the score of the sequence similarity of the nucleic acid sequences in the data, and the sequence information corresponding to all the microbeads in the same micro-well is combined and restored to a single cell / nucleus to obtain the final single-cell sequencing data.

10. A method of single cell sequencing, the method comprising: The method comprises the following steps: The single-cell multi-omics library is constructed by using the single-cell library construction method based on the double-pass penetration type micro-well chip according to any one of claims 1 to 8, and includes any one of the following: a transcriptome and a chromatin accessible genome; The obtained single-cell multi-omics library is sequenced to obtain single-cell transcriptome and chromatin accessibility genome data; The genetic information of the single-cell multi-omics is obtained by using the data processing method according to claim 9, and the genetic information of the single-cell multi-omics is reduced to a single cell according to the genetic information similarity and the nucleic acid molecules of the known base sequence on the multi-well plate and the microbeads.

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