Single-cell enrichment and mixed-sample library construction sequencing methods for large cohort samples

Abstract

The present invention discloses a method for single-cell enrichment and mixed-sample library construction and sequencing of large-scale cohort samples, comprising the following steps: S1, forming single-cell samples from multiple sources; S2, incubating single-cell samples from different sources with biotin antibodies and labeling them with streptavidin-coupled oligonucleotides, and then mixing multiple samples to form a mixed sample; S3, preparing 3' transcriptome cDNA samples and labeled DNA samples; S4, constructing a 3' transcriptome library; S5, constructing a labeled DNA library; S6, merging the 3' transcriptome library and the labeled DNA library for sequencing analysis. The technical methods of single-cell enrichment, streptavidin-coupled oligonucleotide labeling, single-cell transcriptome mixed-sample library construction, sequencing, and analysis provided by the present invention are suitable for large-scale cohort sample research and rare cell population research such as infectious diseases, inflammation, and tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical research, and specifically relates to a method for single-cell enrichment of large cohort samples, avidin antibody incubation, streptavidin-coupled oligonucleotide sample labeling, single-cell transcriptome mixed sample library construction, sequencing, and analysis. Background Art

[0002] Transcriptome sequencing (RNA Sequencing, RNA-seq) refers to the high-throughput sequencing of mRNA produced by a species or a specific cell in a certain functional state. It can not only detect differences in gene expression levels and provide quantitative analysis, but also provide structural analysis, identify variable splicing sites, gene fusions, etc., and does not rely on a reference genome. Traditional transcriptome sequencing (Bulk RNA Sequencing, Bulk RNA-seq) measures the average expression level of each gene in a large cell population. The study of the nature of gene expression is not in-depth enough. The spatiotemporal information of many gene expressions is obscured and cannot reflect the heterogeneity between tissue cells (Li X, Wang CY. From bulk, single-cell to spatial RNA sequencing [J]. Int J Oral Sci, 2021, 13 (1): 36).

[0003] In recent years, single-cell RNA-seq (scRNA-seq) technology has been rapidly developed and can reveal gene expression differences at the single-cell level. scRNA-seq is applied to different types of tissue cells and cell lines of various species (especially humans, mice, etc.), including normal and diseased cells (Kuksin M, Morel D, Aglave M. Applications of single-cell and bulk RNA sequencing in onco-immunology [J]. Eur J Cancer, 2021, 149: 193-210). scRNA-seq can reveal the gene structure and gene expression status of individual cells, reflect the heterogeneity between cells, and play an important role in tumors, developmental biology, microbiology, neuroscience and other fields (Paik DT, Cho S, Tian L, Chang HY, Wu JC [J]. Single-cell RNA sequencing in cardiovascular development, disease and medicine. Nat Rev Cardiol, 2020, 17 (8): 457-473).

[0004] According to the sequence range of transcripts captured by sequencing, scRNA-seq can be mainly divided into full-length transcriptome sequencing technology represented by Smart-seq2, and 3' / 5' transcriptome sequencing technology represented by 10X Genomics. The former breaks the cDNA and sequences all fragments of the transcript. The advantage is that the number of gene detections is higher than the latter, and the full-length information of the transcript can be detected. The disadvantages are low cell throughput, high sequencing costs for single cells, and the detection is not the true full-length transcript. 10XGenomics uses Barcoding and Microfluidics technology, which has advantages in the principles of single-cell separation and amplification. The cell throughput that can be captured is high, but the sequencing cost is very expensive. (Kashima Y,Sakamoto Y,Kaneko K.Single-cell sequencing techniques from individual to multiomicsanalyses[J].ExpMolMed,2020,52:1419-1427;ViethB,Parekh S,ZiegenhainC.Asystematic evaluation of single cell RNA-seq analysis pipelines[J].NatCommun,2019,10(1):4667;Wang X,He Y,Zhang Q,Ren X,Zhang Z.Direct comparative analyzes of 10X Genomics chromium and Smart-seq2[J].Genom Proteom Bioinf, 2021,19(2):253-266)

[0005] In summary, the following defects exist in current single-cell preparation and scRNA-seq library construction and sequencing:

[0006] 1. In comprehensive scRNA-seq analyses, batch effects have been shown to mask biological signals, potentially leading to false-negative or false-positive results. If batch and treatment effects do not completely overlap, the ratio of between-group differences to within-group differences is reduced, thereby reducing the significance of the treatment effect. If batch and treatment effects completely overlap or nearly overlap (orthogonal effects), the batch effect generally amplifies inter-group differences, making it difficult to distinguish between differences caused by the experimental treatment and batch effects.

[0007] Batch effects primarily impact single-cell data by affecting quantification through changes in gene expression. Secondly, they can cause cells that should have been clustered together to be separated into different clusters, affecting the accuracy of cell subpopulation identification and, in turn, downstream analysis. This problem is particularly severe in large cohort studies of infectious diseases, inflammation, or tumors, where multiple samples are collected at different times and sequenced in multiple batches.

[0008] 2. Large cohort studies are the most direct manifestation of epidemiological design and methods in medical big data research. Their strong evidence base and good extrapolation capabilities provide researchers with a more comprehensive and in-depth understanding, avoiding the randomness and bias that can result from small clinical sample sizes. However, single-cell transcriptome sequencing technology is currently expensive to build and sequence, and the sample pre-processing process is complex and lengthy, hindering its widespread application in clinical disease research.

[0009] 3. Currently, scRNA-seq requires high sample preparation requirements before processing. The starting cell count for a single sample must be 105-106, with a viable cell count exceeding 80%, with a recommended viability of over 90%. Single-cell isolation and acquisition from certain tissues can be challenging, and the number and viability of isolated and enriched single cells may not meet current scRNA-seq library construction and sequencing requirements.

[0010] Research on infectious diseases, inflammation, and tumors often involves blood or bone marrow mononuclear cells, NK cells, NKT cells, Treg cells, circulating tumor cells, and tumor TIL cells. The isolation and enrichment of these cells from blood, bone marrow, and tumor tissue samples is challenging, and the number of cells per sample is small. Obtaining sufficient numbers of active single cells is a prerequisite for building high-quality single-cell libraries and obtaining valid sequencing data. Current single-cell isolation, enrichment, and library construction and sequencing solutions cannot address this issue.

[0011] Due to the various shortcomings and defects of current scRNA-seq, it is difficult to apply it to large cohort sample studies and rare cell population studies. Summary of the Invention

[0012] Purpose of the invention: The purpose of the present invention is to address the shortcomings of the existing technology and provide a method for single-cell enrichment, biotin antibody incubation, streptavidin-coupled oligonucleotide sample labeling, and single-cell transcriptome mixed sample library construction and sequencing, which is suitable for large cohort sample studies and rare cell population studies such as infectious diseases, inflammation, and tumors.

[0013] Technical solution: The high-throughput single-cell transcriptome multi-sample mixed library construction and sequencing method of the present invention comprises the following steps:

[0014] S1. Isolate and enrich target cells from blood, bone marrow, or tumor tissues of different sources to form single-cell samples;

[0015] S2. Incubating the single cell samples from multiple sample sources obtained in step S1 with a biotin antibody, labeling each sample with a streptavidin-coupled oligonucleotide, and then mixing the samples from different sources to form a mixed sample;

[0016] S3. Place the mixed sample into the Chromium Controller to obtain GEMs, then perform reverse transcription on the GEMs to obtain a DNA solution. PCR mix, transcriptome cDNA primers, and labeled DNA primers are then added to the resulting DNA product for amplification. The labeled DNA sample and 3' transcriptome cDNA sample are then separated and recovered by agarose gel electrophoresis.

[0017] S4. Use the 3' transcriptome cDNA sample to perform adapter ligation and library sequencing index PCR reaction to construct a 3' transcriptome library;

[0018] S5. Performing library sequencing index PCR reaction using the labeled DNA sample to construct a labeled DNA library;

[0019] S6. Denature and dilute the 3' transcriptome library and the tagged DNA library according to the requirements of the sequencing platform, and combine the 3' transcriptome library and the tagged DNA library for sequencing.

[0020] A further preferred technical solution of the present invention is that after step S1 is completed, the single cell sample that is not to be processed in step S2 in the short term is frozen and the sample is revived before step S2 is performed.

[0021] Preferably, the specific method for freezing the sample is:

[0022] Ⅰ. Pre-cool the cell program cooling box, cryopreservation tubes and cryopreservation solution at 4°C in advance;

[0023] II. Determine the total cell count and cell viability of single-cell samples using a cell counter;

[0024] III. Place the single-cell sample in a centrifuge and centrifuge at 400 × g for 7 min at 4°C. Remove the supernatant.

[0025] IV. Keep the single cell sample on ice and resuspend the cell pellet in culture medium to a cell concentration of 4-20×10 6 cells / mL;

[0026] V. Add an equal volume of pre-freezing solution to make the cell concentration 2-10×10 6 cells / mL and mix gently;

[0027] VI. Aliquot the cell suspension into pre-chilled cryovials and place the cryovials into a pre-chilled cell program cooling box;

[0028] VII. Place the cell program cooling box at -80℃ for more than 4 hours, and then transfer the cryovials to liquid nitrogen for long-term storage.

[0029] Preferably, the specific method for resuscitating the sample is:

[0030] I. Remove the cryovial containing the single-cell sample and immediately thaw it in a 37°C water bath for 2-3 minutes. When it is thawed until only tiny ice crystals remain, remove it from the water bath. In a clean bench, rinse the cryovial with pre-warmed complete growth medium and transfer the thawed single-cell sample to a centrifuge tube using a pipette.

[0031] II. While shaking the centrifuge tube, add the same volume of culture medium as the single-cell sample into the centrifuge tube at a rate of 1 mL / 3-5 seconds. Repeat this dilution cycle for a total of 5 times, waiting 1 minute between additions. Then, place the centrifuge tube in a centrifuge and centrifuge at 400 × g for 7 minutes. Remove the supernatant and resuspend the cell pellet using a pipette.

[0032] III. Add culture medium to the tube again, mix the cells, and count the cells to determine the cell concentration until the cell concentration is 1.8-2.2×10 6 / mL, transfer the suspension in the tube to another centrifuge tube and centrifuge again at 400×g for 7 min, remove the supernatant, add buffer PBS+ into the tube with a wide-mouth pipette and mix by pipetting;

[0033] IV. Rinse the centrifuge tube with PBS+ buffer and transfer the cell suspension to an EP tube. Centrifuge at 400×g for 7 minutes and remove the supernatant. Add PBS+ buffer to the EP tube to obtain a cell suspension with a concentration of 600-1200 cells / μL. Gently mix the cell suspension with a pipette.

[0034] V. The cell suspension was passed through a 70 μm mesh to remove cell clumps and obtain a single-cell suspension. The single-cell suspension was placed on ice for subsequent high-throughput single-cell transcriptome library construction and sequencing.

[0035] Preferably, the single cell sample in step S1 is a tumor tissue TIL cell, circulating tumor cells, blood or bone marrow mononuclear cells, NK, NKT or Treg rare cells in blood or bone marrow, one of which is enriched.

[0036] Preferably, when the single cell sample in step S1 is a tumor tissue TIL cell, the single cell sample preparation method is:

[0037] Ⅰ. Excise fresh tumor tissue, trim away necrotic parts and connective tissue, rinse with PBS+ buffer, transfer to a sterile dish, and cut the tumor tissue into 1-2 mm pieces with surgical scissors. 3 Small pieces of tumor tissue were mixed with digestion solution, transferred to a sterile Erlenmeyer flask with a magnetic bar, and stirred on a magnetic stirrer at 37°C for 1-3 hours. The digested cell suspension was filtered through a 120 μm mesh filter to remove undigested tumor tissue. The digestion solution was a cocktail enzyme solution containing 0.05% collagenase type IV, 0.001% DNase I, and 1500 U / g hyaluronidase type V.

[0038] II. Isolate and collect single cells into a 50 mL centrifuge tube; wash the sterile Erlenmeyer flask twice with digestion solution and transfer all the liquid into the 50 mL centrifuge tube; place the 50 mL centrifuge tube on ice for 10 minutes, centrifuge at 50 × g for 2 minutes, transfer the supernatant to a new 50 mL centrifuge tube, centrifuge again at 400 × g for 7 minutes, discard the supernatant, and add 3 mL of PBS+ buffer to resuspend the cells;

[0039] III. Sequentially add 3 mL of 1.088 Ficoll-Hypaque, an equal volume of 1.075 Ficoll-Hypaque, and the tumor tissue cell suspension to a new centrifuge tube and perform discontinuous density gradient centrifugation at 500 × g for 20 minutes.

[0040] IV. Collect the cell suspension at the interface between the upper and lower Ficoll-Hypaque layers to obtain a TIL-rich cell suspension. Wash the cells twice with PBS+ buffer. After resuspending the cells, filter through a 70 μm mesh to obtain a single-cell suspension of tumor tissue TIL cells.

[0041] Preferably, when the single cell sample in step S1 is a circulating tumor cell, the single cell sample is prepared by:

[0042] I. Add 15 mL of whole blood sample to a test tube, collect cells by centrifugation, and lyse RBCs; add PBS+ buffer to the test tube and mix thoroughly. Centrifuge at 400 × g for 7 minutes at room temperature, and remove the supernatant.

[0043] II. Resuspend the cells in 0.5-1 mL of PBS+ buffer, add 50 μL of enrichment cock-tail antibody per mL of blood sample, mix well, and incubate at room temperature for 5 minutes. The enrichment cock-tail antibody is a biotinylated mixed reagent of anti-human CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin A antibodies.

[0044] III. Add negative selection magnetic beads to the mixture of antibody and cell suspension incubation. Add 50 μL of negative selection magnetic beads per mL of blood sample-derived cells. Add PBS + buffer to make up to 10 mL of sample. Gently pipette up and down 2-3 times to mix. Negative selection magnetic beads are streptavidin-coupled magnetic beads.

[0045] IV. Place the test tube in a magnet and incubate at room temperature for 10 minutes. Pipette the enriched cell suspension into a new test tube.

[0046] V. Repeat steps III to IV and enrich twice to obtain high-purity circulating tumor cells.

[0047] Preferably, when the single cell sample in step S1 is formed from blood or bone marrow mononuclear cells, the single cell sample is prepared by:

[0048] I. Prepare 10 mL of whole blood or bone marrow sample and mononuclear cell separation buffer and equilibrate at room temperature for 30 minutes.

[0049] II. Add 10 mL each of blood or bone marrow and PBS to a 50 mL centrifuge tube and gently pipette until mixed. Add 10-20 mL of mononuclear cell separation buffer to the bottom of a second 50 mL centrifuge tube. Tilt the tube to 45 degrees and pipette 20 mL of the whole blood or bone marrow sample diluted with PBS from the first 50 mL centrifuge tube slowly along the wall and add it to the upper layer of the mononuclear cell separation buffer in the second 50 mL centrifuge tube. Then transfer the second 50 mL centrifuge tube to a centrifuge, set the ascent speed to 1 and the descent speed to 0, and centrifuge at room temperature and 700 × g for 30-40 minutes.

[0050] After centrifugation, pipette the PBMC cells from the second 50 mL centrifuge tube and transfer them to a 15 mL centrifuge tube. Add PBS+ buffer to a final volume of 14 mL. Invert the cell suspension 3-5 times to mix thoroughly. Transfer the 15 mL centrifuge tube to a centrifuge and centrifuge at 400 × g for 7 minutes at room temperature. Remove the centrifuge tube from the centrifuge and observe the cell pellet after centrifugation. If red color is present in the cell pellet, perform red blood cell lysis as follows:

[0051] a. Use a wide-mouth pipette tip to discard the supernatant in a 15 mL centrifuge tube and add 3 mL of red blood cell lysis buffer. Incubate at 4°C and time the red blood cell lysis for 3-5 minutes.

[0052] b. Add 5-7 mL of pre-chilled PBS+ buffer and gently invert to mix. Transfer the 15 mL tube to a centrifuge and centrifuge at 400 × g for 7 minutes at room temperature.

[0053] c. Observe the cell pellet after centrifugation again. If visible red blood cell pellets are still mixed in the cell suspension, repeat steps a to b. The red blood cell lysis time during the repeated process shall not exceed 3 minutes.

[0054] IV. After centrifugation, remove the centrifuge tube containing the cell pellet from the centrifuge, discard the supernatant, and aspirate the remaining liquid at the tube mouth; add 1 mL of pre-chilled PBS+ buffer and gently pipette to resuspend the cells, then add PBS+ buffer to a final volume of 10 mL. Place the 15 mL centrifuge tube back into the centrifuge and centrifuge at room temperature at 400 × g for 7 minutes to collect the cell pellet;

[0055] V. Repeat step IV 1-2 times to wash the cells and remove background. If a large number of platelets are present in the cell pellet under a microscope, resuspend the cell pellet in PBS+ buffer and centrifuge at room temperature at 300×g for 5-7 minutes to remove the platelets.

[0056] VI. Resuspend the cell pellet with 100-200 μL of PBS+ buffer, pass the cell suspension through a 70 μm mesh to remove cell clusters, and obtain a blood mononuclear cell sample in a single cell suspension state.

[0057] Preferably, when the single cell sample in step S1 is formed from NK, NKT or Treg rare cells in blood or bone marrow, the single cell sample is prepared by:

[0058] I. Prepare mononuclear cells from blood or bone marrow and wash twice with PBS+ buffer to remove platelets. Adjust the concentration of PBMC suspension to 1×10 8 / mL. If cell clumps are present, thoroughly pipette to mix or filter through a 70μm mesh to obtain a single-cell suspension. Place the required number of cells in a 5mL test tube, not exceeding 2×10 8 indivual;

[0059] II. Add 20 μL of enrichment cocktail antibody per 100 μL of cells, vortex to mix the cells and antibodies evenly, and incubate at room temperature for 7-12 minutes; add PBS+ buffer to a volume of 4 mL to wash the cells, then centrifuge at 400 × g for 7 minutes, discard the supernatant, resuspend in PBS+ buffer, and mix thoroughly by pipetting to reduce the cell suspension to a single cell state; the cocktail antibody is a mixed reagent of biotin-labeled anti-human CD3 antibody, CD4 antibody, CD14 antibody, CD19 antibody, CD20 antibody, CD66b antibody, CD123 antibody, CD235a antibody, and HLA-DR antibody;

[0060] III. Vortex to thoroughly resuspend the cells. Add 10 μL of negative selection magnetic beads per 100 μL of cells, mix thoroughly, and incubate at room temperature for 5 minutes. Add PBS+ buffer to a volume of 2.5 mL and mix by pipetting with a 1 mL pipette. The negative selection magnetic beads should be streptavidin-coupled.

[0061] IV. Insert the test tube into the magnet and incubate at room temperature for 5 min. Remove the magnet and pour the supernatant into a 15 mL conical tube. Hold the tube upside down for 1 s and then return it to an upright position.

[0062] Ⅴ. Remove the test tube from the magnet and repeat steps III-IV once more. The two cell suspensions pooled in the 15 mL conical tube are the NK cells obtained by negative selection.

[0063] Preferably, the specific method of incubating the single cell sample with biotin antibodies in step S2 is:

[0064] Ⅰ. Add multiple single cell samples from different sources into corresponding 1.5 mL low binding tubes and resuspend each sample to 1-2 × 10 6 cell;

[0065] II. Add 10 μL Fc blocking reagent and incubate at 4°C for 10 min.

[0066] III. Add 0.5 μg of biotin-conjugated antibody to each low-binding tube, incubate at 4°C for 15 min, and then wash three times with 1 mL of staining buffer. Centrifuge the low-binding tubes at 400 × g for 7 min at 4°C.

[0067] The specific method for labeling each sample with streptavidin-coupled oligonucleotides is as follows:

[0068] IV. Add a different and unique streptavidin-conjugated oligonucleotide to each low-binding tube and incubate at 4°C for 20 minutes.

[0069] V. Wash the cells three times with 1 mL of PBS+ buffer, then centrifuge the low-binding tube at 400 × g for 7 min at 4°C.

[0070] The specific method for mixing the samples to form a mixed sample is as follows:

[0071] VI. Combine the samples from 3-5 low-binding tubes in equal proportions into 1 mL of PBS+ buffer and centrifuge at 400 × g for 7 min at 4°C to obtain several mixed samples.

[0072] VII. Resuspend the mixed samples of each group in PBS+ to a cell concentration of 500-1200 cells / μL;

[0073] VIII. Filter the cells with a 70 μm mesh to obtain a single-cell suspension. Analyze the cell concentration and cell viability of the obtained single-cell suspension on a counter, and select mixed samples with a cell viability higher than 90%.

[0074] Preferably, when the single cell sample in step 1 is formed by TIL cells from tumor tissue, in step S2-III, the biotin-conjugated antibody is a biotin-conjugated CD3 antibody;

[0075] When the single cell sample in step 1 is formed from circulating tumor cells, in step S2-III, the biotin-conjugated antibody is a biotin-conjugated pan CK antibody, including CK8, CK18, and CK19;

[0076] When the single cell sample in step 1 is formed from blood or bone marrow mononuclear cells, in step S2-III, the biotin-conjugated antibody is a biotin-conjugated CD45 antibody;

[0077] When the single cell sample in step 1 is formed from NK or NKT rare cells in blood or bone marrow, in step S2-III, the biotin-conjugated antibody is a biotin-conjugated CD56 antibody;

[0078] When the single cell sample in step 1 is formed from rare Treg cells in blood or bone marrow, in step S2-III, the biotin-conjugated antibody is a biotin-conjugated CD4 antibody or a CD25 antibody.

[0079] Preferably, the sequence of the streptavidin-coupled oligonucleotide added in step IV is:

[0080] SEQ ID NO.1TCTATCTGCTGCAAT

[0081] SEQ ID NO.2GTGTCCATACCGCTA

[0082] SEQ ID NO.3ATCCTATGGCACGGC

[0083] SEQ ID NO.4CTGGGACTAATAGCT

[0084] SEQ ID NO.5CACGTAGATGTCAAT

[0085] SEQ ID NO.6TATATCCACCCGCTC

[0086] SEQ ID NO.7TATGTGACCACGTGA

[0087] SEQ ID NO.8TCTCCGAATCATTTA

[0088] SEQ ID NO.9ACCGCTATTCGGCGA

[0089] SEQ ID NO.10TACTTACGTGCATGC

[0090] SEQ ID NO.11GTCTAGTCATTCGCT

[0091] SEQ ID NO. 12TCATTGCCTATAGCG.

[0092] Preferably, the specific method of step S3 is:

[0093] I. Dispense 70 μL of each mixed sample into the bottom center of each well in the first row of the chip. Slowly aspirate 50 μL of gel beads and dispense them into each well in the second row, waiting 30 seconds. Dispense 45 μL of oil into each well in the third row, place the sample in the Chromium Controller, and run the program to obtain GEMs.

[0094] II. Transfer the GEMs into thin-walled 8-tube PCR strips and perform reverse transcription at 53°C for 45 minutes and 85°C for 5 minutes. Add 125 μL of recovery agent to each GEM sample at room temperature, let it stand for 2 minutes, and discard 125 μL of recovery agent from the bottom of the tube.

[0095] III. Add the obtained DNA to the equilibrated adsorption column equipped with a collection tube, let it stand at room temperature for 3 minutes, and centrifuge at 12,000 × g for 2 minutes, discarding the waste liquid; add the rinse solution to the adsorption column, let it stand for 4 minutes, and centrifuge again at 12,000 × g for 2 minutes, discarding the waste liquid;

[0096] IV. Centrifuge at 12,000 × g for 3 minutes to remove any remaining rinse solution from the column. Then air-dry the column at room temperature. Install a new collection tube on the column and drip nuclease-free water onto the center of the column membrane. Allow to stand at room temperature for 3 minutes. Centrifuge again at 12,000 × g for 3 minutes to collect the DNA solution and determine its concentration.

[0097] V. The obtained DNA product was added to PCR mix, transcriptome cDNA primers, and labeled DNA primers for amplification reaction. The amplification reaction conditions were as follows: Step 1: 98°C, 3 min; Step 2: 98°C, 15 s, 63°C, 20 s, 72°C, 1 min, 12 cycles; Step 3: 72°C, 1 min;

[0098] VI. Separate and recover the labeled DNA and 3' transcriptome cDNA by agarose gel electrophoresis. Using 350 bp as the cutoff, recover smaller fragments as labeled DNA and larger fragments as 3' transcriptome cDNA. Cut the target DNA band from the agarose gel and place it in a centrifuge tube. Weigh the weight, then add an equal volume of recovery buffer to the gel, incubate at 50°C in a water bath for 12 minutes, and continue mixing to ensure dissolution.

[0099] VII. Repeat steps III to IV with the resulting solution to obtain labeled DNA samples and 3' transcriptome cDNA samples.

[0100] Preferably, the specific method for constructing the 3' transcriptome library in step S4 is:

[0101] I. Prepare 10 μL of enzyme digestion buffer, 5 μL of nucleic acid fragmentation enzyme, 25 μL of EB buffer, and 10 μL of 3' transcriptome cDNA sample, and perform fragmentation and blunt-end reaction procedures. The enzyme digestion reaction conditions are 30°C for 8 minutes; the blunt-end reaction conditions are 70°C for 1 hour. After completion, store at 4°C. Then, perform agarose gel electrophoresis on the sample and recover the 250-600bp nucleic acid band to obtain the 3' transcriptome cDNA.

[0102] II. Prepare 20 μL of ligation reaction buffer, 20 μL of adapter nucleotides, and 50 μL of 3' transcriptome cDNA sample, and perform adapter ligation reaction on ice at 20°C for 15 minutes. Store at 4°C after completion. Then, perform agarose gel electrophoresis on the sample to recover 250-600 bp nucleic acid bands and 3' transcriptome cDNA.

[0103] III. Prepare 50 μL of high-fidelity PCR Mix, 10 μL of index primers, and 30 μL of 3' transcriptome cDNA sample for library sequencing index PCR reaction. The reaction conditions are as follows: Step 1: 97°C, 50 s; Step 2: 97°C, 25 s; Step 2: 55°C, 25 s; Step 3: 72°C, 1 min; Store at 4°C after completion; Then, perform agarose gel electrophoresis on the sample to recover the 250-600 bp nucleic acid band and the 3' transcriptome cDNA.

[0104] IV. Analyze the 3' transcriptome library on a DNA bioanalyzer. After sample dilution, run 1 μL of sample for QC and quantification.

[0105] The specific method of step S5 for constructing the labeled DNA library is:

[0106] I. Prepare 50 μL high-fidelity PCR Mix, 10 μL index primers, and 40 μL labeled DNA sample for library sequencing index PCR reaction. The reaction conditions are as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 20 s; 64°C, 30 s; 70°C, 25 s; cycle 10-13 times; Step 3: 72°C, 5 min; store at 4°C; then perform agarose gel electrophoresis on the sample to recover the 170-200 bp nucleic acid band and the labeled DNA.

[0107] II. Analyze the labeled DNA gene library on a DNA bioanalyzer. After sample dilution, run 1 μL of sample for QC and quantitative analysis.

[0108] Preferably, the specific method of step S6 is:

[0109] I. After quantifying and normalizing the constructed tagged 3' transcriptome library and tagged DNA library, denature and dilute the 3' gene expression library and tagged DNA library according to the requirements of the sequencing platform;

[0110] II. Based on the differences in cell number and per-cell read depth requirements between each library, the 3' gene expression library and the tagged DNA library were combined for sequencing.

[0111] Beneficial effects: (1) The present invention provides a 6-80 sample mixed library construction sequencing technology, which can label single-cell samples from different patients in a large cohort study, or from multiple batches and multiple time points, with different oligonucleotides and then mix them to build a library. All samples can be sequenced on the same machine, which significantly reduces the cost of single-cell transcriptome sequencing and promotes the application of this technology in large cohort studies.

[0112] (2) The single-cell isolation technology of the present invention is optimized for precious samples, ensuring minimal cell loss, high cell yield, and viability exceeding 90%. Using a cell-safe layering solution or a mild modified digestion solution, while achieving efficient single-cell preparation, it fully ensures the effective maintenance of cell sample surface antigen information and protects the original transcriptional expression profile of the cells.

[0113] (3) The rare cell population enrichment technology provided by the present invention uses negative selection to label and remove non-target cells to obtain target cells. This avoids direct labeling of target cells, thereby reducing the possibility of cell activation. The advantages of this solution are that it can retain the target cell population to the greatest extent, and can keep the target cells in their original state without stimulation, and the sorting speed is fast.

[0114] (4) The single-cell sample freezing and recovery technology provided by the present invention overcomes the defects that samples must be put on the machine immediately after collection, and the temporal and spatial differences in sequencing lead to significant batch effects. After single-cell separation and enrichment, the sample can be stored at -80°C or liquid nitrogen for more than one year. The frozen cells from cell lines and primary samples show RNA molecule integrity similar to that of freshly prepared cells, and the gene expression profile has no obvious changes. In the case of freezing, the storage and transportation of samples can be achieved, so that large queues of samples can be centrally processed and mixed to build a library before sequencing. The present invention also provides a technical solution for the recovery of frozen single-cell samples, and the proportion of active cells after recovery can reach more than 90%. At the same time, centralized sample processing can also save the time of multiple library constructions, reduce experimental costs and labor costs.

[0115] (5) The present invention proposes a cell separation and enrichment scheme and reagents optimized for precious samples, which minimizes cell loss, and the resulting single cell yield is high, and the purity and activity meet the requirements for single-cell library construction and sequencing. The present invention also discloses single-cell separation and enrichment, sample freezing and recovery technology, so that large cohorts of samples can be sequenced and analyzed in the same batch. More importantly, the biotin antibody incubation and streptavidin-coupled oligonucleotide labeling of single-cell samples from different sources and then mixed library construction and sequencing technology disclosed in the present invention greatly reduce the cost of single-cell transcriptome sequencing, and solve the problem that the number of rare cell populations is too small and a single sample cannot meet the requirements for library construction and sequencing. In a broad sense, the method and optimized reagent formula disclosed in the present invention can be used to prepare single-cell transcriptome sample pre-treatment and mixed sample library construction kits, related compositions or chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 Images of tumor-infiltrating lymphocytes after enrichment. Compared with other separation and enrichment methods (A), this method enriched TIL cells (B) with higher yield, better cell morphology, and better activity.

[0117] Figure 2 Single-cell samples were cryopreserved and thawed, followed by trypan blue staining and counting. Compared with the conventional cryopreservation and thawing method (A), this protocol yielded cells (B) with higher yields, better morphology, and better viability.

[0118] Figure 3 This protocol produces quality control and yield plots of cDNA amplification products. High cDNA yield and a concentrated product size distribution are achieved.

[0119] Figure 4 Other protocols were used to generate cDNA amplification product quality control and yield plots. The cDNA product fragments were disorganized and contained multiple peaks, which affected subsequent library construction results.

[0120] Figure 5 This protocol was used to prepare cDNA amplification products for library construction and QC analysis. This protocol produced high yield and uniform fragments.

[0121] Figure 6 QC analysis of cDNA amplification products prepared with other protocols. Libraries prepared with other protocols may have uneven fragments and contain artifacts, which may affect subsequent sequencing.

[0122] Figure 7 Flow cytometry analysis of circulating tumor cells before and after enrichment. The purity of circulating tumor cells after enrichment reached 96%.

[0123] Figure 8 Flow cytometry analysis of blood mononuclear cells (PBMCs). The percentage of PBMC single cells is as high as 98.8%, and the percentage of live cells is 98.1%.

[0124] Figure 9 Schematic diagram of the process for enriching NK cells from blood.

[0125] Figure 10 QC analysis of labeled DNA and 3'cDNA sequencing. The quality control analysis results show that the base content distribution of the samples measured by this protocol is normal (A) and the sequencing quality is high (B).

[0126] Figure 11 Distribution of sequencing data. Violin plots show the number of UMIs per cell (A) and the number of different oligonucleotide markers per cell (B).

[0127] Figure 12 Heat map of labeled DNA from samples of different origins.

[0128] Figure 13 Cluster diagram of tumor-infiltrating lymphocytes (TIL cells) with different characteristics.

[0129] Figure 14 Cluster diagram of tumor-infiltrating lymphocytes (TIL cells) from different sample sources.

[0130] Figure 15 Cluster diagram of cells from different sample sources in PBMC mixed sample library construction and sequencing.

[0131] Figure 16 Clustering diagram of different types of immune cells sequenced from PBMC mixed sample library.

[0132] Figure 17 Volcano plot of differentially expressed genes in NK cells from the blood of patients and healthy individuals.

[0133] Figure 18 Heat map of differentially expressed genes in circulating tumor cells (CTCs) of different cancer types.

[0134] Figure 19 Flowchart for single-cell enrichment and transcriptome pooled library construction and sequencing. DETAILED DESCRIPTION

[0135] The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the embodiments.

[0136] Example 1: A method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples. In this example, tumor tissue TIL cells are used as samples, including the following steps:

[0137] S1. Enrichment of TIL cells in tumor tissue:

[0138] Ⅰ. Excise fresh tumor tissue, trim away necrotic parts and connective tissue, rinse with PBS+ buffer, transfer to a sterile dish, and cut the tumor tissue into 1-2 mm pieces with surgical scissors. 3 Small pieces of tumor tissue were mixed with digestion solution, transferred to a sterile Erlenmeyer flask with a magnetic bar, and stirred on a magnetic stirrer at 37°C for 1-3 hours. The digested cell suspension was filtered through a 120 μm mesh filter to remove undigested tumor tissue. The digestion solution was a cocktail enzyme solution containing 0.05% collagenase type IV, 0.001% DNase I, and 1500 U / g hyaluronidase type V.

[0139] II. Isolate and collect single cells into a 50 mL centrifuge tube; wash the sterile Erlenmeyer flask twice with digestion solution and transfer all the liquid into the 50 mL centrifuge tube; place the 50 mL centrifuge tube on ice for 10 minutes, centrifuge at 50 × g for 2 minutes, transfer the supernatant to a new 50 mL centrifuge tube, centrifuge again at 400 × g for 7 minutes, discard the supernatant, and add 3 mL of PBS+ buffer to resuspend the cells;

[0140] III. Sequentially add 3 mL of 1.088 Ficoll-Hypaque, an equal volume of 1.075 Ficoll-Hypaque, and the tumor tissue cell suspension to a new centrifuge tube and perform discontinuous density gradient centrifugation at 500 × g for 20 minutes.

[0141] IV. Collect the cell suspension at the interface between the upper and lower Ficoll-Hypaque layers to obtain a cell suspension rich in TILs. Wash the cells twice with PBS+ buffer, resuspend the cells, and filter through a 70 μm mesh to obtain a single cell suspension of tumor tissue TIL cells. Figure 1 As shown, compared with other separation and enrichment methods, compared with other separation and enrichment methods (A), the TIL cells (B) enriched by this method have high yield, good cell morphology and activity.

[0142] The enriched TIL cells can be added to freezing medium and stored in a liquid nitrogen tank, or incubated on ice to label single cell samples for subsequent library construction and sequencing.

[0143] S2. Cryopreservation of isolated and enriched single cells:

[0144] Ⅰ. Pre-cool the cell program cooling box, cryopreservation tubes and cryopreservation solution at 4°C in advance;

[0145] II. Determine the total cell count and cell viability of single-cell samples using a cell counter;

[0146] III. Place the single-cell sample in a centrifuge and centrifuge at 400 × g for 7 min at 4°C. Remove the supernatant.

[0147] IV. Keep the single cell sample on ice and resuspend the cell pellet in culture medium to a cell concentration of 4-20×10 6 cells / mL;

[0148] V. Add an equal volume of pre-freezing solution to make the cell concentration 2-10×10 6 cells / mL and mix gently;

[0149] VI. Aliquot the cell suspension into pre-chilled cryovials and place the cryovials into a pre-chilled cell program cooling box;

[0150] VII. Place the cell program cooling box at -80℃ for more than 4 hours, and then transfer the cryovials to liquid nitrogen for long-term storage.

[0151] S3. Isolation and enrichment to obtain single cell recovery:

[0152] I. Remove the cryovial containing the single-cell sample and immediately thaw it in a 37°C water bath for 2-3 minutes. When it is thawed until only tiny ice crystals remain, remove it from the water bath. In a clean bench, rinse the cryovial with pre-warmed complete growth medium and transfer the thawed single-cell sample to a centrifuge tube using a pipette.

[0153] II. While shaking the centrifuge tube, add the same volume of culture medium as the single-cell sample into the centrifuge tube at a rate of 1 mL / 3-5 seconds. Repeat this dilution cycle for a total of 5 times, waiting 1 minute between additions. Then, place the centrifuge tube in a centrifuge and centrifuge at 400 × g for 7 minutes. Remove the supernatant and resuspend the cell pellet using a pipette.

[0154] III. Add culture medium to the tube again, mix the cells, and count the cells to determine the cell concentration until the cell concentration is 1.8-2.2×10 6 / mL, transfer the suspension in the tube to another centrifuge tube and centrifuge again at 400×g for 7 min, remove the supernatant, add buffer PBS+ into the tube with a wide-mouth pipette and mix by pipetting;

[0155] IV. Rinse the centrifuge tube with PBS+ buffer and transfer the cell suspension to an EP tube. Centrifuge the EP tube at 400 × g for 7 minutes and remove the supernatant. Add PBS+ buffer to the EP tube to obtain a cell suspension with a concentration of 600-1200 cells / μL. Gently mix the cell suspension with a pipette.

[0156] V. The cell suspension was passed through a 70 μm mesh to remove cell clumps and obtain a single-cell suspension. The single-cell suspension was placed on ice for subsequent high-throughput single-cell transcriptome library construction and sequencing.

[0157] like Figure 2 As shown, compared with the conventional cryopreservation-thawing method (A), the cells obtained with this protocol (B) have higher yields, better cell morphology, and better viability. After thawing, the cell viability reaches over 90%, which meets the requirements for single-cell transcriptome library sequencing.

[0158] S4. Incubation of multiple samples with biotin antibodies and indirect labeling of samples with streptavidin-coupled oligonucleotides:

[0159] Ⅰ. Add single cell samples from different sources into corresponding 1.5 mL low binding tubes and resuspend each sample to 1-2 × 10 6 cell;

[0160] II. Add 10 μL Fc blocking reagent and incubate at 4°C for 10 min.

[0161] III. Add 0.5 μg of biotin-conjugated CD3 antibody to each low-binding tube, incubate at 4°C for 15 min, and then wash three times with 1 mL of staining buffer. Centrifuge the low-binding tubes at 400 × g for 7 min at 4°C.

[0162] IV. Add a different and unique streptavidin-coupled oligonucleotide to each low-binding tube and incubate at 4°C for 20 minutes; the oligonucleotide barcode streptavidin sequence is:

[0163] SEQ ID NO.1TCTATCTGCTGCAAT

[0164] SEQ ID NO.2GTGTCCATACCGCTA

[0165] SEQ ID NO.3ATCCTATGGCACGGC

[0166] SEQ ID NO.4CTGGGACTAATAGCT

[0167] SEQ ID NO.5CACGTAGATGTCAAT

[0168] SEQ ID NO.6TATATCCACCCGCTC

[0169] SEQ ID NO.7TATGTGACCACGTGA

[0170] SEQ ID NO.8TCTCCGAATCATTTA

[0171] SEQ ID NO.9ACCGCTATTCGGCGA

[0172] SEQ ID NO.10TACTTACGTGCATGC

[0173] SEQ ID NO.11GTCTAGTCATTCGCT

[0174] SEQ ID NO. 12TCATTGCCTATAGCG.

[0175] V. Wash the cells three times with 1 mL of PBS+ buffer, then centrifuge the low-binding tube at 400 × g for 7 min at 4°C.

[0176] VI. Combine the samples from 3-5 low-binding tubes in equal proportions into 1 mL of PBS+ buffer and centrifuge at 400 × g for 7 min at 4°C to obtain several mixed samples.

[0177] VII. Resuspend the mixed samples from each group in PBS+ to a cell concentration of 500-1200 cells / μL;

[0178] VIII. Filter the cells with a 70 μm mesh to obtain a single-cell suspension. Analyze the cell concentration and cell viability of the obtained single-cell suspension on a counter, and select mixed samples with a cell viability higher than 90%.

[0179] S5. Preparation and isolation of labeled DNA and 3' transcriptome cDNA:

[0180] I. Dispense 70 μL of each mixed sample into the bottom center of each well in the first row of the chip. Slowly aspirate 50 μL of gel beads and dispense them into each well in the second row, waiting 30 seconds. Dispense 45 μL of oil into each well in the third row, place the sample in the Chromium Controller, and run the program to obtain GEMs.

[0181] II. Transfer the GEMs into thin-walled 8-tube PCR strips and perform reverse transcription at 53°C for 45 minutes and 85°C for 5 minutes. Add 125 μL of recovery agent to each GEM sample at room temperature, let it stand for 2 minutes, and discard 125 μL of recovery agent from the bottom of the tube.

[0182] III. Add the obtained DNA to the equilibrated adsorption column equipped with a collection tube, let it stand at room temperature for 3 minutes, and centrifuge at 12,000 × g for 2 minutes, discarding the waste liquid; add the rinse solution to the adsorption column, let it stand for 4 minutes, and centrifuge again at 12,000 × g for 2 minutes, discarding the waste liquid;

[0183] IV. Centrifuge at 12,000 × g for 3 minutes to remove any remaining rinse solution from the column. Then air-dry the column at room temperature. Install a new collection tube on the column and drip nuclease-free water onto the center of the column membrane. Allow to stand at room temperature for 3 minutes. Centrifuge again at 12,000 × g for 3 minutes to collect the DNA solution and determine its concentration.

[0184] V. The obtained DNA product was added to PCR mix, transcriptome cDNA primers, and labeled DNA primers for amplification reaction. The amplification reaction conditions were as follows: Step 1: 98°C, 3 min; Step 2: 98°C, 15 s, 63°C, 20 s, 72°C, 1 min, 12 cycles; Step 3: 72°C, 1 min;

[0185] VI. Separate and recover the labeled DNA and 3' transcriptome cDNA by agarose gel electrophoresis. Using 350 bp as the cutoff, recover smaller fragments as labeled DNA and larger fragments as 3' transcriptome cDNA. Cut the target DNA band from the agarose gel and place it in a centrifuge tube. Weigh the weight, then add an equal volume of recovery buffer to the gel, incubate at 50°C in a water bath for 12 minutes, and continue mixing to ensure dissolution.

[0186] VII. Repeat steps III to IV with the resulting solution to obtain labeled DNA samples and 3' transcriptome cDNA samples.

[0187] Run 1 μL of sample on a DNA bioanalyzer for QC and quantification. Figure 3 As shown in Figure 2, this protocol produces high yield cDNA and a concentrated product size distribution. Figure 4 As shown in the figure, the fragments of cDNA amplification products prepared by other schemes are distributed in a disorderly manner, with multiple impurity peaks, which affects the subsequent library construction results.

[0188] S6, 3' transcriptome library construction

[0189] I. Prepare 10 μL of enzyme digestion buffer, 5 μL of fragmentation enzyme, 25 μL of EB buffer, and 10 μL of 3' transcriptome cDNA sample, and perform fragmentation and blunt-end reaction procedures. The enzyme digestion reaction conditions are 30°C for 8 minutes; the blunt-end reaction conditions are 70°C for 1 hour. After completion, store at 4°C. Then, perform agarose gel electrophoresis on the sample, recover the 250-600bp nucleic acid band, and recover the 3' transcriptome cDNA.

[0190] II. Prepare 20 μL of ligation reaction buffer, 20 μL of adapter nucleotides, and 50 μL of 3' transcriptome cDNA sample, and perform adapter ligation reaction on ice at 20°C for 15 minutes. Store at 4°C after completion. Then, perform agarose gel electrophoresis on the sample to recover 250-600 bp nucleic acid bands and 3' transcriptome cDNA.

[0191] III. Prepare 50 μL of high-fidelity PCR Mix, 10 μL of index primers, and 30 μL of 3' transcriptome cDNA sample for library sequencing index PCR reaction. The reaction conditions are as follows: Step 1: 97°C for 50 seconds; Step 2: 97°C for 25 seconds; Step 3: 55°C for 25 seconds; Step 4: 70°C for 25 seconds; Cycle 11-15 times; Step 3: 72°C for 1 minute. Store at 4°C. Then, perform agarose gel electrophoresis on the sample to recover the 250-600 bp nucleic acid band and the 3' transcriptome cDNA.

[0192] IV. Analyze the 3' transcriptome library on a DNA bioanalyzer. After sample dilution, run 1 μL of sample for QC and quantitative analysis. Figure 5 As shown in Figure 2, the library prepared by this protocol has high yield and uniform fragments. Figure 6 As shown in the figure, the library fragments prepared by other schemes are uneven and contain mixed peaks, which affects subsequent sequencing.

[0193] S7. Construction of labeled DNA library:

[0194] I. Prepare 50 μL high-fidelity PCR Mix, 10 μL index primers, and 40 μL labeled DNA sample for library sequencing index PCR reaction. The reaction conditions are as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 20 s; 64°C, 30 s; 70°C, 25 s; cycle 10-13 times; Step 3: 72°C, 5 min; store at 4°C; then perform agarose gel electrophoresis on the sample to recover the 170-200 bp nucleic acid band and the labeled DNA.

[0195] II. Analyze the labeled DNA gene library on a DNA bioanalyzer. After sample dilution, run 1 μL of sample for QC and quantitative analysis.

[0196] S8, 3' transcriptome library and tagged DNA library mixed sequencing:

[0197] I. After quantifying and normalizing the constructed 3' transcriptome library and tagged DNA library, denature and dilute them according to the requirements of the sequencing platform;

[0198] II. Based on the differences in cell number and per-cell read depth requirements between each library, the 3' transcriptome library and the tagged DNA library were combined for sequencing.

[0199] Example 2: In this example, circulating tumor cells were used as samples.

[0200] A method for single-cell enrichment and pooled library construction for large cohort samples, comprising the following steps:

[0201] S1. Circulating tumor cell enrichment:

[0202] I. Add 15 mL of whole blood sample to a test tube, collect cells by centrifugation, and lyse RBCs; add PBS+ buffer to the test tube and mix thoroughly. Centrifuge at 400 × g for 7 minutes at room temperature, and remove the supernatant.

[0203] II. Resuspend the cells in 0.5-1 mL of PBS+ buffer, add 50 μL of enrichment cock-tail antibody per mL of blood sample, mix well, and incubate at room temperature for 5 minutes. The enrichment cock-tail antibody is a mixed reagent of biotin-labeled anti-CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin A antibodies.

[0204] III. Add negative selection magnetic beads to the mixture of antibody and cell suspension incubation. Add 50 μL of negative selection magnetic beads per mL of blood sample-derived cells. Add PBS + buffer to make up to 10 mL of sample. Gently pipette up and down 2-3 times to mix. Negative selection magnetic beads are streptavidin-coupled magnetic beads.

[0205] IV. Place the test tube in a magnet and incubate at room temperature for 10 minutes. Pipette the enriched cell suspension into a new test tube.

[0206] V. Repeat steps III to IV and enrich twice to obtain high-purity circulating tumor cells.

[0207] The enriched circulating tumor cells can be added to freezing solution and stored in a liquid nitrogen tank, or incubated on ice to label single cell samples for subsequent library construction and sequencing.

[0208] like Figure 7 As shown, this protocol can enrich high-yield CTC cells, and the CTC purity can reach more than 90%.

[0209] S2-S3 are the same as in Example 1.

[0210] S4. The only difference from Example 1 is that the biotin-conjugated antibody added to the low-binding tube in this example is pan CK antibody, including CK8 / CK18 / CK19.

[0211] S5-S8 are the same as in Example 1.

[0212] Example 3: In this example, blood mononuclear cells were used as samples.

[0213] A method for single-cell enrichment and pooled library construction for large cohort samples, comprising the following steps:

[0214] S1. Isolation of blood mononuclear cells:

[0215] I. Prepare 10 mL of whole blood sample and mononuclear cell separation solution and equilibrate at room temperature for 30 minutes.

[0216] II. Add 10 mL each of blood and PBS to a 50 mL centrifuge tube and gently pipette until mixed. Add 10-20 mL of mononuclear cell separation solution to the bottom of a second 50 mL centrifuge tube. Tilt the tube to 45 degrees and pipette 20 mL of PBS-diluted blood from the first 50 mL centrifuge tube slowly along the wall and add it to the mononuclear cell separation solution layer in the second 50 mL centrifuge tube. Transfer the second 50 mL centrifuge tube to a centrifuge, set the ascent speed to 1 and the descent speed to 0, and centrifuge at room temperature and 700 × g for 30-40 minutes.

[0217] After centrifugation, pipette the PBMC cells from the second 50 mL centrifuge tube and transfer them to a 15 mL centrifuge tube. Add PBS+ buffer to a final volume of 14 mL. Invert the cell suspension 3-5 times to mix thoroughly. Transfer the 15 mL centrifuge tube to a centrifuge and centrifuge at 400 × g for 7 minutes at room temperature. Remove the centrifuge tube from the centrifuge and observe the cell pellet after centrifugation. If red color is present in the cell pellet, perform red blood cell lysis as follows:

[0218] a. Use a wide-mouth pipette tip to discard the supernatant in a 15 mL centrifuge tube and add 3 mL of red blood cell lysis buffer. Incubate at 4°C and time the red blood cell lysis for 3-5 minutes.

[0219] b. Add 5-7 mL of pre-chilled PBS+ buffer and gently invert to mix. Transfer the 15 mL tube to a centrifuge and centrifuge at 400 × g for 7 minutes at room temperature.

[0220] c. Observe the cell pellet after centrifugation again. If visible red blood cell pellets are still mixed in the cell suspension, repeat steps a to b. The red blood cell lysis time during the repeated process shall not exceed 3 minutes.

[0221] IV. After centrifugation, remove the centrifuge tube containing the cell pellet from the centrifuge, discard the supernatant, and aspirate the remaining liquid at the tube mouth; add 1 mL of pre-chilled PBS+ buffer and gently pipette to resuspend the cells, then add PBS+ buffer to a final volume of 10 mL. Place the 15 mL centrifuge tube back into the centrifuge and centrifuge at room temperature at 400 × g for 7 minutes to collect the cell pellet;

[0222] V. Repeat step IV 1-2 times to wash the cells and remove background. If a large number of platelets are present in the cell pellet under a microscope, resuspend the cell pellet in PBS+ buffer and centrifuge at room temperature at 300×g for 5-7 minutes to remove the platelets.

[0223] VI. Resuspend the cell pellet with 100-200 μL of PBS+ buffer, pass the cell suspension through a 70 μm mesh to remove cell clusters, and obtain a human peripheral blood mononuclear cell sample in a single cell suspension state.

[0224] The isolated mononuclear cells (PBMCs) can be added to freezing solution and stored in a liquid nitrogen tank, or incubated on ice to label single cell samples for subsequent library construction and sequencing.

[0225] like Figure 8 As shown in the figure, the peripheral blood mononuclear cells (PBMCs) isolated by this protocol can reach 1-2×10 8 cells / mL, the single cell ratio is greater than 98%, and the cell activity is above 98%.

[0226] S2-S3 are the same as in Example 1.

[0227] S4. The only difference from Example 1 is that the biotin-conjugated antibody added to the low-binding tube in this example is biotin-conjugated CD45 antibody.

[0228] S5-S8 are the same as in Example 1.

[0229] Example 4: In this example, NK cells in blood were used as samples.

[0230] A method for single-cell enrichment and pooled library construction for large cohort samples, comprising the following steps:

[0231] S1. Blood NK cell enrichment, the process is as follows Figure 9 As shown:

[0232] Mononuclear cells were isolated from blood and washed twice with PBS+ buffer to remove platelets. The concentration of PBMC cell suspension was adjusted to 1×10 8 / mL. When cell clumps are present, thoroughly pipette to mix or filter through a 70μm mesh to obtain a single-cell suspension. Place the required number of cells in a 5mL round-bottom polystyrene tube, with the number not exceeding 2×10 8 indivual;

[0233] II. Add 20 μL of enrichment cocktail antibody per 100 μL of cells, vortex to mix the cells and antibodies evenly, and incubate at room temperature for 7-12 minutes; add PBS+ buffer to a small test tube with a liquid volume of 4 mL to wash the cells, then centrifuge at 400×g for 7 minutes, discard the supernatant, resuspend in PBS+ buffer, and mix thoroughly by pipetting to a single cell state; the cocktail antibody is a mixed reagent of biotin-labeled anti-CD3 antibody, CD4 antibody, CD14 antibody, CD19 antibody, CD20 antibody, CD66b antibody, CD123 antibody, CD235a antibody, and HLA-DR antibody;

[0234] III. Vortex to thoroughly resuspend the cells. Add 10 μL of negative selection magnetic beads per 100 μL of cells, mix thoroughly, and incubate at room temperature for 5 minutes. Add PBS+ buffer to a volume of 2.5 mL in the small test tube and mix by pipetting with a 1 mL pipette. The negative selection magnetic beads should be streptavidin-coupled.

[0235] IV. Insert the test tube into the magnet and incubate at room temperature for 5 min. Remove the magnet and pour the supernatant into a 15 mL conical tube. Hold the tube upside down for 1 s and then return it to an upright position.

[0236] Ⅴ. Remove the test tube from the magnet and repeat steps III-IV once more. The two cell suspensions pooled in the 15 mL conical tube are the NK cells obtained by negative selection.

[0237] The enriched NK cells can be added to freezing solution and stored in a liquid nitrogen tank, or incubated on ice to label single cell samples for subsequent library construction and sequencing.

[0238] Negative selection magnetic beads are streptavidin-coupled magnetic beads that bind to biotin-labeled antibodies. This protocol utilizes negative selection to isolate and enrich rare NK cell populations from blood samples, with the enriched NK cell percentage reaching over 90%.

[0239] S2-S3 are the same as in Example 1.

[0240] S4. The only difference from Example 1 is that the biotin-conjugated antibody added to the low-binding tube in this example is biotin-conjugated CD56 antibody.

[0241] S5-S8 are the same as in Example 1.

[0242] Analyze the sequencing data obtained in the above example:

[0243] The QC analysis of the sequencing results showed that the sequencing results were stable and the base content distribution of the samples was normal ( Figure 10 A), high-quality sequencing data ( Figure 10 B).

[0244] Preprocessing of mixed sequencing data of 3' transcriptome library and tagged DNA library:

[0245] Ⅰ. Use fastq-dump software to process SRA data and obtain fastq format files. Use software such as CellRanger in Linux system to construct data. Load the UMI matrix and labeled DNA count matrix in R Studio, and select sample data according to different samples of the labeled DNA count matrix. Select cell barcodes that can be detected by both "RNA" and "labeled DNA", and filter the UMI matrix and labeled DNA count matrix into subsets with joint cell barcodes. Set the new RNA count data as a Seurat object, and add the previously processed labeled DNA data as another experimental condition different from "RNA", so that the data can be analyzed under the labeled DNA experimental condition. The violin plot of the sequencing data shows the number of UMIs per cell (11A) and the number of different oligonucleotide markers per cell (11B). The violin plot visualizes the basic distribution of sequencing data, showing that the number of genes in the sequenced cells is normal.

[0246] Ⅱ. Use the Seurat function Demux() to assign individual cells back to their sample sources based on their marker DNA levels, and save the results as the "metadata" of the Seurat object. Use the ncells parameter to subsample the cells and use the DNAHeatmap() function to create a heat map of marker DNA from different sample sources, such as Figure 12 Figure 3 shows the normalized marker DNA values in different samples, indicating that mixed samples can be clearly distinguished.

[0247] III. Further quality control and normalization of the demultiplexed count matrix data were performed. Genomic alignment and expression results were used to filter the cells detected by sequencing, removing cells with low gene counts and a high proportion of mitochondrial genes. The number of filtered cells was counted and the corresponding expression matrix was generated. The NormalizeData function was used to normalize the gene expression values in each cell and identify genes (DEGs) that were highly variable between cells. The ScaleData function was used to scale the data for downstream dimensionality reduction analysis.

[0248] Clinical multi-sample mixed library sequencing data analysis and visualization:

[0249] I. Principal Component Analysis (PCA) and t-SNE Cluster Analysis. Principal component analysis (PCA) was performed on the scaled data using the "RunPCA" function. Each principal component was scored using the "JackStraw" and "ScoreJackStraw" functions. The scores were visualized using the "JackStrawPlot" function. Each principal component was visualized using the "ElbowPlot" function, and the corresponding principal components were selected for cluster analysis. Clustered cells were visualized using nonlinear dimensionality reduction (UMAP). t-SNE cluster analysis was performed using the "RunUMAP" function, and visualization was performed using the "UMAPPlot" function.

[0250] like Figure 13-14 As shown in the figure, in this example, single-cell transcriptome sequencing data was obtained after processing a mixed sample of tumor-infiltrating lymphocytes (TIL) to build a library and sequence it. 15 clusters were obtained through dimensionality reduction clustering, and different sample sources could be identified. Figure 15-16 As shown in the figure, sequencing data from a mixed peripheral blood mononuclear cell (PBMC) sample library was constructed. Dimensionality reduction clustering can identify different sample sources and analyze different immune cell types and functional states. This library construction and sequencing solution provides reliable sequencing data, effectively separating single-cell data from different sample sources and reflecting the true biological characteristics of the samples.

[0251] Ⅱ. A volcano plot is a type of scatter plot that combines the statistical significance measure and the amplitude of change in statistical tests, thereby helping to quickly and intuitively identify those data points (genes, etc.) with large amplitudes of change and statistical significance. The volcano plot can conveniently and intuitively display the distribution of differential gene expression between samples. The horizontal axis is represented by log2 (foldchange), and genes with greater differences are distributed at both ends. The vertical axis is represented by -log10 (p value), which is the negative logarithm of the T-test significance P value. Generally, the larger the fold difference, the more significant the T-test of genes, so the data points in the upper left and upper right corners are often more meaningful for biological research. In the study of infectious diseases, inflammation and other diseases, various immune cell populations are often involved, such as Figure 17 As shown in the figure, in this case, human peripheral blood NK cells were enriched, and single-cell transcriptome sequencing data was obtained after library construction and sequencing of the mixed samples. It can be seen that the expression levels of multiple chemokine ligands (CCL) and heat shock protein family members in patients with inflammatory conditions were significantly increased.

[0252] Ⅲ. Heatmap can use color changes to reflect the data information in a two-dimensional matrix or table. It can intuitively represent the size of sequencing data with defined color depths. Data are often clustered based on abundance similarity between species or samples as needed, and the clustered data are represented on a heatmap. High-abundance and low-abundance samples can be clustered in blocks, and the similarities and differences of multiple samples can be reflected through color gradients and similarity levels. R language vegan package, vegdist and hclust perform distance calculation and cluster analysis; distance algorithm: chao, clustering method: complete. Figure 18 As shown in this example, single-cell transcriptome sequencing data was obtained after processing a mixed circulating tumor cell (CTC) sample library and sequencing. Nine clusters were obtained by identifying differentially expressed genes and performing dimensionality reduction clustering. CTC clustering from different cancer sources showed different characteristic genes, which has important guiding significance for the use of CTCs in early cancer diagnosis.

[0253] The present invention provides a technical method for single cell enrichment, sample labeling with avidin antibodies and streptavidin-coupled oligonucleotides, single cell transcriptome library construction and sequencing, and analysis. The present invention is applicable to large cohort sample studies and rare cell population studies for infectious diseases, inflammation, tumors, etc. The process overview of the present invention is shown in the figure below. Figure 19 shown.

[0254] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims. Sequence Listing <120> Single-cell enrichment and mixed-sample library construction sequencing methods for large cohort samples <140> 2022105306323 <141> 2022-05-16 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <400> 1 tctatctgct gcaat 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <400> 2 gtgtccatac cgcta 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <400> 3 atcctatggc acggc 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <400> 4 ctgggactaa tagct 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <400> 5 cacgtagatg tcaat 15 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <400> 6 tatatccacc cgctc 15 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <400> 7 tatgtgacca cgtga 15 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <400> 8 tctccgaatc attta 15 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <400> 9 accgctattc ggcga 15 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <400> 10 tacttacgtg catgc 15 <210> 11 <211> 15 <212> DNA <213> Artificial Sequence <400> 11 gtctagtcat tcgct 15 <210> 12 <211> 15 <212> DNA <213> Artificial Sequence <400> 12 tcattgccta tagcg 15

Claims

1. A method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples, characterized by: The steps include: S1. Isolate and enrich target cells from blood, bone marrow, or tumor tissue samples from different sources to form single-cell samples; S2, incubating the single cell samples from multiple sample sources obtained in step S1 with a biotin antibody, labeling each sample with a streptavidin-coupled oligonucleotide, and then mixing the samples from different sources to form a mixed sample; S3. Place the mixed sample into the Chromium Controller to obtain GEMs, then perform reverse transcription on the GEMs to obtain a DNA solution. PCR mix, transcriptome cDNA primers, and labeled DNA primers are then added to the obtained DNA product for amplification. The labeled DNA sample and 3' transcriptome cDNA sample are then separated and recovered by agarose gel electrophoresis. S4. Use 3' transcriptome cDNA samples to perform adapter ligation and library sequencing index PCR reaction to construct a 3' transcriptome library; S5. Perform library sequencing index PCR reaction using the labeled DNA sample to construct a labeled DNA library; S6. Denature and dilute the 3' transcriptome library and the tagged DNA library according to the requirements of the sequencing platform, and combine the 3' transcriptome library and the tagged DNA library for sequencing; The single cell sample in step S1 is formed by enrichment of one of tumor tissue TIL cells, circulating tumor cells, blood or bone marrow mononuclear cells, or NK rare cells in blood or bone marrow; In step S2, the single cell sample obtained in step S1 is incubated with a biotin-conjugated antibody: When the single-cell sample is formed by TIL cells from tumor tissue, the biotin-conjugated antibody is a biotin-conjugated CD3 antibody; When the single-cell sample is formed from circulating tumor cells, the biotin-conjugated antibody is a biotin-conjugated pan CK antibody, which includes CK8, CK18, and CK19; When the single cell sample is formed from blood or bone marrow mononuclear cells, the biotin-conjugated antibody is a biotin-conjugated CD45 antibody; When the single cell sample is formed from NK rare cells in blood or bone marrow, the biotin-conjugated antibody is a biotin-conjugated CD56 antibody; The sequences of the streptavidin-coupled oligonucleotides added in step S2 are SEQ ID NO. 1 to SEQ ID NO.

12.

2. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: After step S1 is completed, the single cell sample that will not be processed in step S2 in the short term is frozen and revived before step S2; The specific method for freezing samples is as follows: Ⅰ. Pre-cool the cell program cooling box, cryopreservation tubes and cryopreservation solution at 4°C in advance; II. Determine the total cell count and cell viability of single-cell samples using a cell counter; III. Place the single-cell sample in a centrifuge and centrifuge at 400 × g for 7 min at 4°C. Remove the supernatant. IV. Keep the single cell sample on ice and resuspend the cell pellet in culture medium to a cell concentration of 4-20×10 6 cells / mL; V. Add an equal volume of pre-freezing solution to make the cell concentration 2-10×10 6 cells / mL and mix gently; VI. Aliquot the cell suspension into pre-chilled cryovials and place the cryovials into a pre-chilled cell program cooling box; VII. Place the cell program cooling box at -80℃ for more than 4 hours, and then transfer the cryovials to liquid nitrogen for long-term storage.

3. The method for single-cell enrichment and mixed-sample library construction and sequencing of large cohort samples according to claim 2, characterized in that: The specific method for sample recovery is: I. Remove the cryovial containing the single-cell sample and immediately thaw it in a 37°C water bath for 2-3 minutes. When only tiny ice crystals remain, remove it from the water bath. In a clean hood, rinse the cryovial with pre-warmed complete growth medium and transfer the thawed single-cell sample to a centrifuge tube using a pipette. II. While shaking the tube, add the same volume of culture medium as the single-cell sample into the tube at a rate of 1 mL / 3-5 s. Repeat this dilution cycle for a total of 5 times, waiting 1 minute between additions. Then, place the tube in a centrifuge and centrifuge at 400 × g for 7 minutes. Remove the supernatant and resuspend the cell pellet using a pipette. III. Add culture medium to the tube again, mix the cells, and count the cells to determine the cell concentration until the cell concentration is 1.8-2.2×10 6 / mL, transfer the suspension in the tube to another centrifuge tube and centrifuge again at 400×g for 7 min, remove the supernatant, add buffer PBS+ into the tube with a wide-mouth pipette and mix thoroughly by pipetting; IV. Rinse the centrifuge tube with PBS+ buffer and transfer the cell suspension to a new EP tube. Centrifuge at 400 × g for 7 min and remove the supernatant. Add PBS+ buffer to the EP tube to obtain a cell suspension with a concentration of 600-1200 cells / μL. Gently mix the cell suspension with a pipette. V. The cell suspension was passed through a 70 µm mesh to remove cell clumps and obtain a single-cell suspension. The single-cell suspension was placed on ice for subsequent high-throughput single-cell transcriptome library construction and sequencing.

4. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: When the single cell sample in step S1 is a tumor tissue TIL cell, the preparation method of the single cell sample is: Ⅰ. Excise fresh tumor tissue, trim away necrotic parts and connective tissue, rinse with PBS+ buffer, transfer to a sterile dish, and cut the tumor tissue into 1-2 mm pieces with surgical scissors. 3 Small pieces of tumor tissue were mixed with digestion solution, transferred to a sterile Erlenmeyer flask with a magnetic bar, and stirred on a magnetic stirrer at 37°C for 1-3 hours. The digested cell suspension was filtered through a 120 μm mesh filter to remove undigested tumor tissue. The digestion solution was a cocktail enzyme solution containing 0.05% collagenase type IV, 0.001% DNase I, and 1500 U / g hyaluronidase type V. II. Isolate and collect single cells into a 50 mL centrifuge tube; wash the sterile Erlenmeyer flask twice with digestion solution and transfer all the liquid to the 50 mL centrifuge tube; place the 50 mL centrifuge tube on ice for 10 minutes, centrifuge at 50 × g for 2 minutes, transfer the supernatant to a new 50 mL centrifuge tube, centrifuge at 400 × g for 7 minutes, discard the supernatant, and add 3 mL of PBS+ buffer to resuspend the cells; III. Sequentially add 3 mL of 1.088 Ficoll-Hypaque, an equal volume of 1.075 Ficoll-Hypaque, and the tumor tissue cell suspension to a new centrifuge tube and perform discontinuous density gradient centrifugation at 500 × g for 20 min. IV. Collect the cell suspension at the interface between the upper and lower Ficoll-Hypaque layers to obtain a TIL-rich cell suspension. Wash the cells twice with PBS+ buffer. After resuspending the cells, filter them through a 70μm mesh to obtain a single-cell suspension of tumor tissue TIL cells.

5. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: When the single cell sample in step S1 is formed from blood or bone marrow mononuclear cells, the single cell sample preparation method is: I. Prepare 10 mL of whole blood or bone marrow sample and mononuclear cell isolation buffer and equilibrate at room temperature for 30 minutes. II. Add 10 mL each of whole blood or bone marrow sample and PBS to a 50 mL centrifuge tube (No. 1) and gently pipette until thoroughly mixed. Add 10-20 mL of mononuclear cell separation buffer to the bottom of a second 50 mL centrifuge tube. Tilt the tube to 45 degrees and pipette 20 mL of the whole blood or bone marrow sample diluted with PBS from the first 50 mL centrifuge tube along the wall and slowly add it to the mononuclear cell separation buffer layer in the second 50 mL centrifuge tube. Transfer the second 50 mL centrifuge tube to a centrifuge, set the acceleration to 1 and the deceleration to 0, and centrifuge at 700 × g for 30-40 min at room temperature. After centrifugation, pipette the PBMC layer from the second 50 mL centrifuge tube and transfer it to a 15 mL centrifuge tube. Add PBS+ buffer to a final volume of 14 mL. Invert the cell suspension 3-5 times to mix thoroughly. Transfer the 15 mL centrifuge tube to a centrifuge and centrifuge at 400 × g for 7 min at room temperature. Remove the centrifuge tube from the centrifuge and observe the cell pellet after centrifugation. If there is red in the cell pellet, perform red blood cell lysis. The specific method is as follows: a. Use a wide-mouth pipette tip to discard the supernatant in a 15 mL centrifuge tube and add 3 mL of red blood cell lysis buffer. Incubate at 4°C and time the red blood cell lysis for 3-5 minutes. b. Add 5-7 mL of pre-chilled PBS+ buffer and gently invert to mix. Transfer the 15 mL tube to a centrifuge and centrifuge at 400 × g for 7 min at room temperature. c. Observe the cell pellet after centrifugation again. If visible red blood cell pellets are still mixed in the cell suspension, repeat steps a and b. The red blood cell lysis time should not exceed 3 minutes during the repeated process. IV. After centrifugation, remove the centrifuge tube containing the cell pellet from the centrifuge, discard the supernatant, and aspirate the remaining liquid at the top of the tube. Add 1 mL of pre-chilled PBS+ buffer and gently pipette to resuspend the cells. Then add PBS+ buffer to a final volume of 10 mL. Place the 15 mL centrifuge tube back into the centrifuge and centrifuge at room temperature at 400 × g for 7 minutes to collect the cell pellet. V. Repeat step IV 1-2 times to wash the cells to remove background. If a large number of platelets are present in the cell pellet under a microscope, resuspend the cell pellet in PBS+ buffer and centrifuge at 300 × g for 5-7 minutes at room temperature to remove the platelets. VI. Resuspend the cell pellet in 100-200 µL of PBS+ buffer and pass the cell suspension through a 70 µm mesh to remove cell clumps to obtain a single-cell suspension of blood mononuclear cells.

6. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: When the single cell sample in step S1 is a circulating tumor cell, the single cell sample preparation method is: I. Add 15 mL of whole blood sample to a test tube, collect cells by centrifugation, and lyse RBCs. Add PBS+ buffer to the test tube and mix thoroughly. Centrifuge at 400 × g for 7 minutes at room temperature and remove the supernatant. II. Resuspend the cells in 0.5-1 mL of PBS+ buffer. Add 50 µL of enrichment antibody cocktail per mL of blood sample, mix thoroughly, and incubate at room temperature for 5 minutes. The enrichment antibody cocktail is a biotinylated mixture of anti-human CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin A antibodies. III. Add negative selection magnetic beads to the mixture of antibody and cell suspension incubation, adding 50 µL of negative selection magnetic beads per mL of blood sample-derived cells. Add PBS + buffer to make up to 10 mL of sample, and gently pipette up and down 2-3 times to mix. Negative selection magnetic beads are streptavidin-coupled magnetic beads. IV. Place the test tube in a magnet and incubate at room temperature for 10 minutes. Pipette the enriched cell suspension into a new test tube. Ⅴ. Repeat steps III to IV and enrich twice to obtain high-purity circulating tumor cells.

7. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: When the single cell sample in step S1 is a rare NK cell in blood or bone marrow, the single cell sample preparation method is: I. Isolate and prepare mononuclear cells from blood or bone marrow and wash twice with PBS+ buffer to remove platelets; adjust the concentration of PBMC cell suspension to 1×10 8 / mL. If cell clumps are present, thoroughly pipette to mix or filter through a 70 µm mesh to obtain a single-cell suspension. Place the required number of cells in a 5 mL test tube, not exceeding 2 × 10 8 indivual; II. Add 20 µL of enrichment antibody cocktail per 100 µL of cells, vortex to mix the cells and antibodies, and incubate at room temperature for 7-12 minutes. Add PBS+ buffer to a volume of 4 mL to wash the cells, then centrifuge at 400 × g for 7 minutes. Discard the supernatant and resuspend the cells in PBS+ buffer. Mix thoroughly by pipetting to reduce the cell suspension to a single cell state. The cocktail antibody is a mixture of biotinylated anti-human CD3, CD4, CD14, CD19, CD20, CD66b, CD123, CD235a, and HLA-DR antibodies. III. Vortex to thoroughly resuspend the cells. Add 10 µL of negative selection magnetic beads per 100 µL of cells, mix thoroughly, and incubate at room temperature for 5 min. Add PBS+ buffer to a volume of 2.5 mL and mix by pipetting with a 1 mL pipette. The negative selection magnetic beads should be streptavidin-coupled. IV. Insert the test tube into the magnet and incubate at room temperature for 5 min. Remove the magnet and pour the supernatant into a 15 mL conical tube. Hold the tube upside down for 1 s and then return it to an upright position. Ⅴ. Remove the test tube from the magnet and repeat steps III-IV once more. The two cell suspensions pooled in the 15 mL conical tube are the NK cells obtained by negative selection.

8. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: The specific method for incubating the single cell sample with biotin antibodies in step S2 is as follows: Ⅰ. Add multiple single cell samples from different sources into corresponding 1.5 mL low binding tubes and resuspend each sample to 1-2×10 6 cell; II. Add 10 µL of Fc blocking reagent and incubate at 4°C for 10 min. III. Add 0.5 µg of biotin-conjugated antibody to each low-binding tube, incubate at 4°C for 15 min, and then wash three times with 1 mL of staining buffer. Centrifuge the low-binding tubes at 400 × g for 7 min at 4°C. The specific method for labeling each sample with streptavidin-coupled oligonucleotides is as follows: IV. Add a different and unique streptavidin-conjugated oligonucleotide to each low-binding tube and incubate at 4°C for 20 minutes. V. Wash the cells three times with 1 mL of PBS+ buffer, then centrifuge the low-binding tube at 400 × g for 7 min at 4°C. The specific method for mixing the samples to form a mixed sample is as follows: VI. Combine the samples from 3-5 low-binding tubes in equal proportions into 1 mL of PBS+ buffer and centrifuge at 400 × g for 7 min at 4°C to obtain several mixed samples. VII. Resuspend the mixed samples from each group in PBS+ to a cell concentration of 500-1200 cells / µL. VIII. Filter the cells with a 70 µm mesh to obtain a single-cell suspension. Analyze the cell concentration and cell viability of the obtained single-cell suspension on a counter, and select mixed samples with cell viability higher than 90%.

9. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 1, characterized in that: The specific method of step S3 is: I. Dispense 70 µL of each mixed sample into the bottom center of each well in the first row of the chip. Slowly aspirate 50 µL of gel beads and dispense them into each well in the second row, waiting 30 seconds. Dispense 45 µL of oil into each well in the third row, place the chip in the Chromium Controller, and run the program to obtain GEMs. II. Transfer the GEMs into thin-walled 8-tube PCR strips and perform reverse transcription at 53°C for 45 min and 85°C for 5 min. Add 125 µL of recovery agent to each GEM sample at room temperature, let it stand for 2 min, and discard 125 µL of recovery agent by aspirating from the bottom of the tube. III. Add the obtained DNA to the equilibrated adsorption column equipped with a collection tube and place it at room temperature for 3 minutes. The column was centrifuged at 12,000 × g for 2 minutes, and the waste liquid was discarded. The rinse solution was added to the adsorption column, allowed to stand for 4 minutes, and centrifuged again at 12,000 × g for 2 minutes, and the waste liquid was discarded. IV. Centrifuge at 12,000 × g for 3 minutes to remove any remaining rinse solution from the column. Then air-dry the column at room temperature. Install a new collection tube on the column and drip nuclease-free water onto the center of the column membrane. Allow to stand at room temperature for 3 minutes. Centrifuge again at 12,000 × g for 3 minutes to collect the DNA solution and determine its concentration. V. Add the obtained DNA product to the PCR mix, transcriptome cDNA primers, and labeled DNA primers for amplification. Amplification reaction conditions: Step 1: 98°C, 3 min; Step 2: 98°C, 15 s, 63°C, 20 s, 72°C, 1 min, 12 cycles; Step 3: 72°C, 1 min; VI. Separate and recover the labeled DNA and 3' transcriptome cDNA by agarose gel electrophoresis. Using 350 bp as the cutoff, the smaller fragments are recovered as labeled DNA, and the larger fragments are recovered as 3' transcriptome cDNA. Cut the target DNA band from the agarose gel and place it in a centrifuge tube. Weigh it, then add an equal volume of recovery buffer to the gel. Incubate in a 50°C water bath for 12 minutes, mixing continuously to ensure dissolution. VII. Repeat steps III to IV with the resulting solution to obtain labeled DNA samples and 3' transcriptome cDNA samples.

10. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 9, characterized in that: Step S4: The specific method for constructing the 3' transcriptome library is as follows: I. Prepare 10 µL of enzyme digestion buffer, 5 µL of fragmentase, 25 µL of EB buffer, and 10 µL of 3' transcriptome cDNA sample. Perform fragmentation and blunt-end reactions. The enzyme digestion reaction conditions are 30°C for 8 minutes; the blunt-end reaction conditions are 70°C for 1 hour. After completion, store at 4°C. Then, perform agarose gel electrophoresis on the sample to recover the 250-600 bp nucleic acid band and the 3' transcriptome cDNA. II. Prepare 20 µL of ligation buffer, 20 µL of adapter nucleotides, and 50 µL of 3' transcriptome cDNA sample. Perform adapter ligation on ice at 20°C for 15 minutes. Store at 4°C. Then, perform agarose gel electrophoresis on the sample to recover 250-600 bp nucleic acid bands and 3' transcriptome cDNA. III. Prepare 50 µL of high-fidelity PCR Mix, 10 µL of index primers, and 30 µL of 3' transcriptome cDNA sample for library sequencing index PCR reaction. The reaction conditions are as follows: Step 1: 97°C for 50 s; Step 2: 97°C for 25 s; Step 3: 55°C for 25 s; Step 4: 70°C for 25 s; Cycle 11-15 times; Step 3: 72°C for 1 min. Store at 4°C. Then, perform agarose gel electrophoresis on the sample to recover the 250-600 bp nucleic acid band and the 3' transcriptome cDNA. IV. Analyze the 3' transcriptome library on a DNA bioanalyzer. After sample dilution, run 1 µL of sample for QC and quantification. The specific method of step S5 for constructing the labeled DNA library is: I. Prepare 50 μL high-fidelity PCR Mix, 10 μL index primers, and 40 μL labeled DNA sample for library sequencing index PCR reaction. The reaction conditions are as follows: Step 1: 95°C, 3 min; Step 2: 95°C for 20 s; 64°C for 30 s; 70°C for 25 s; cycle 10-13 times; Step 3: 72°C for 5 min; store at 4°C; then perform agarose gel electrophoresis on the sample to recover the 170-200 bp nucleic acid band and the labeled DNA; II. Analyze the labeled DNA gene library on a DNA bioanalyzer. After sample dilution, run 1 µL of the sample for QC and quantification.

11. The method for single-cell enrichment and mixed-sample library construction and sequencing of a large cohort of samples according to claim 10, characterized in that: The specific method of step S6 is: I. After quantifying and normalizing the constructed 3' transcriptome library and tagged DNA library, denature and dilute the 3' gene expression library and tagged DNA library according to the requirements of the sequencing platform; II. Based on the differences in cell number and per-cell read depth requirements between each library, the 3' transcriptome library and the tagged DNA library were combined for sequencing.

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