Quality control product kit for single cell sequencing and single cell sequencing method

Abstract

The invention discloses a quality control product kit for single cell sequencing and a single cell sequencing method. In the application, it is found for the first time that the CD16 + human peripheral blood mononuclear cell quality control product as a single cell sequencing quality control standard product can exclude problem data in a data analysis stage after single cell sequencing, so that researchers can obtain experimental data completely consistent with that in the real world.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection, and in particular to a quality control kit for single-cell sequencing and a single-cell sequencing method. Background Art

[0002] Standards are substances that are used for calibration, verification or quality control, and have known purity and properties. In the fields of chemical analysis, biological experiments, and drug research, standards are used as benchmarks to ensure the accuracy and consistency of experimental results. Standards are characterized by high purity and known chemical properties, so they are widely used in scientific research, drug development, quality control and other fields.

[0003] Single-cell transcriptome sequencing technology is to perform high-throughput sequencing analysis of the transcriptome at the level of a single cell, which allows researchers to obtain key data that would often be obscured by RNA-seq methods of a large number of cells, such as rare or new cell types, and thus explore gene expression diversity at the single-cell level. This technology can achieve large-scale single-cell transcriptome sequencing, and has the advantages of high cell throughput, high cell capture rate, and short project cycle. It is widely used in studies such as cell heterogeneity, immune cell population detection, and construction of cell maps. It depicts the state of a single cell in the body and the direct relationship between cells and single cells at the genetic level, and is showing an irreplaceable role in basic scientific research and new drug mechanism research. However, there are some problems in the experimental process of single-cell transcriptome sequencing technology that may affect the final data quality, such as cell activity, fragmentation rate, agglomeration rate, total cell volume, etc. At the same time, in addition to the quality of the sample itself, the quality of the relevant reagents on the machine, the deviation of the instrument operation, etc. will also lead to the unreliability of the final single-cell data. At present, there is no good way to overcome the above problems in the field of single-cell sequencing. Once related problems occur, they will be directly attributed to the failure of sample preparation.

[0004] Therefore, the field needs to find effective means to overcome the quality issues of data in single-cell sequencing. Summary of the invention

[0005] The object of the present invention is to provide a quality control kit for single-cell sequencing.

[0006] Another object of the present invention is to provide a use of CD16+ human peripheral blood mononuclear cells.

[0007] To solve the above technical problems, the first aspect of the present invention provides a quality control kit for single-cell sequencing, comprising a plurality of individually packaged CD16+ human peripheral blood mononuclear cell suspensions, wherein the number of cells contained in the CD16+ human peripheral blood mononuclear cell suspensions is known.

[0008] In some preferred embodiments, the kit includes 5 separately packaged CD16+ human peripheral blood mononuclear cell suspensions with known cell numbers, and the cell number of each CD16+ human peripheral blood mononuclear cell suspension is different.

[0009] In some preferred embodiments, the kit further comprises a blank reagent that does not contain CD16+ human peripheral blood mononuclear cells.

[0010] In some preferred embodiments, the kit includes 5 separately packaged CD16+ human peripheral blood monocyte suspensions, wherein the number of CD16+ human peripheral blood monocytes contained in the 5 CD16+ human peripheral blood monocyte suspensions is 200, 500, 1000, 3000 and 5000, respectively.

[0011] In some preferred embodiments, the CD16+ human peripheral blood mononuclear cell suspension comprises: a known number of CD16+ human peripheral blood mononuclear cells and a culture medium, wherein the culture medium is a DMEM culture medium containing 0.04% BSA.

[0012] In some preferred embodiments, the kit comprises the following components:

[0013] name composition Blank Reagent 1 mL of DMEM medium containing 0.04% BSA Standard 1 200 CD16+ human peripheral blood mononuclear cells and 1 mL of DMEM medium containing 0.04% BSA Standard 2 500 CD16+ human peripheral blood mononuclear cells and 1 mL of DMEM medium containing 0.04% BSA Standard 3 1000 CD16+ human peripheral blood mononuclear cells and 1 mL of 0.04% BSA DMEM medium Standard 4 3000 CD16+ human peripheral blood mononuclear cells and 1 mL of DMEM medium containing 0.04% BSA Standard 5 5000 CD16+ human peripheral blood mononuclear cells and 1 mL of DMEM medium containing 0.04% BSA

[0014] The second aspect of the present invention provides a use of CD16+ human peripheral blood mononuclear cells as a quality control product for single-cell sequencing.

[0015] The third aspect of the present invention provides a method for preparing CD16+ human peripheral blood mononuclear cells, comprising the steps of:

[0016] S1: Obtain human peripheral blood mononuclear cells;

[0017] S2: CD16+ monocytes are obtained by sorting.

[0018] In some preferred embodiments, the method of obtaining human peripheral blood mononuclear cells comprises the steps of:

[0019] S11: Obtain a blood sample and filter out impurities therein through a cell sieve;

[0020] S12: Dilute the filtered blood sample with a diluent, and then add an appropriate amount of Ficoll;

[0021] S13: Centrifugation to layer the blood sample with Ficoll and collect mononuclear cells;

[0022] S14: Cultivate the obtained monocytes in culture medium to obtain.

[0023] In some preferred embodiments, the centrifugation to separate the blood sample from the Ficoll layer and collect the mononuclear cells comprises the steps of:

[0024] The blood sample and Ficoll were separated into four layers by centrifugation, and the cells in the second layer were aspirated.

[0025] In some preferred embodiments, the diluent is DPBS.

[0026] In some preferred embodiments, the culturing of the obtained monocytes in a culture medium comprises the steps of:

[0027] The mononuclear cells are cultured in 4 ml of DMEM medium containing 0.04% BSA, and then centrifuged to enrich the mononuclear cells. Preferably, the mononuclear cells are centrifuged at 4° C. for 5 min, and the centrifuge speed is 500×g.

[0028] In some preferred embodiments, the centrifugal enrichment of the monocytes further includes a step of washing the monocytes: taking the centrifugally enriched monocytes, adding 7 ml of DMEM medium containing 0.04% BSA, and centrifuging at a low speed to wash the monocytes. Preferably, the monocytes are centrifuged at 4°C and a centrifuge speed of 500×g for 5 minutes, and the supernatant is discarded to obtain the washed monocytes.

[0029] In some preferred embodiments, in S2, the monocytes are sorted using CD16 positive magnetic beads to obtain CD16+ monocytes.

[0030] In some preferred embodiments, the CD16+ monocytes are resuspended in 1 ml of DMEM medium containing 0.04% BSA and then centrifuged to obtain a CD16+ monocyte suspension, which is a CD16+ human peripheral blood monocyte quality control product.

[0031] A fourth aspect of the present invention provides a single cell sequencing method, the method comprising the steps of:

[0032] Add CD16+ human peripheral blood mononuclear cell quality control to the samples to be sequenced for testing.

[0033] In some preferred embodiments, the CD16+ human peripheral blood mononuclear cell quality control product contains CD16+ human peripheral blood mononuclear cells prepared by the method described in the third aspect of the present invention.

[0034] In some preferred embodiments, the kit described in the first aspect of the present invention is used to detect a sample to be sequenced, comprising the steps of:

[0035] Take 6 equal amounts of the sample to be tested, add blank reagent and standards 1-5 respectively, mix well and then perform sequencing analysis.

[0036] Compared with the prior art, the present invention has at least the following advantages:

[0037] (1) The present invention discovers for the first time that the use of CD16+ human peripheral blood mononuclear cell quality control products as single-cell sequencing quality control standards can eliminate problematic data in the data analysis stage after single-cell sequencing, allowing researchers to obtain experimental data that is completely consistent with the real world.

[0038] (2) The RSC cell suspension prepared by the method of the present invention contains no impurities, no agglomerates, no fragments, no clumps larger than 10 μm and other granular impurities, the cell viability is 97.82%, and the total number of cells is 2 million, which meets the requirements of the MoZhuo single-cell transcriptome sequencing platform for single-cell transcriptome sequencing.

[0039] (3) The present invention uses the above-mentioned RSC cells to develop a single-cell sequencing method, using RSC cells without standard kit addition as a control, and adding 200, 500, 1000, 3000, and 5000 standard kit cells in different proportions to detect the final data results to determine the optimal input amount of standard cells. The optimal concentration of added standard cells determined in this way can be used to detect the quality of samples that were previously difficult to detect, as well as the quality of related reagents on the machine and the deviation of instrument operation. The problems that affect the final data play a quality control role, which is convenient for researchers to better trace the causes of poor sample quality in the experiment, so as to better optimize the overall process of the experiment later.

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0042] Figure 1 This is the existing flow chart of monocyte separation by Ficoll method;

[0043] Figure 2 This is the CD16 fluorescence image of the RSC cells finally obtained under bright field and fluorescent staining microscope;

[0044] Figure 3 This is the final cellranger graph of monocytes in the high viability sample without the addition of standard kit cells;

[0045] Figure 4This is the final cellranger graph of RSC of the high-viability sample with 500 standard kit cells added;

[0046] Figure 5 This is the final cellranger graph of RSC of the high-viability sample with 1000 standard kit cells added;

[0047] Figure 6 This is the final cellranger graph of RSC in which 2000 standard kit cells were added to the high-viability sample;

[0048] Figure 7 This is the final cellranger graph of RSC in which 3000 standard kit cells were added to the high viability sample;

[0049] Figure 8 This is the final cellranger graph of RSC of the high viability sample with 5000 standard kit cells added;

[0050] Fig. 9 This is the final cellranger graph of RSC of the low-viability sample with 1000 standard kit cells added;

[0051] Fig.10 This is the final cellranger graph of monocytes in which 1000 standard kit cells were added to the high viability sample;

[0052] Fig.11 This is the final cellranger graph of monocytes in the low-viability sample without the addition of standard kit cells;

[0053] Fig.12 This is the final cellranger graph of monocytes with low viability sample added with 1000 standard kit cells. DETAILED DESCRIPTION

[0054] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0055] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0056] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0057] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0058] Example 1: Preparation of Standards

[0059] In this example, monocytes are isolated from PBMCs prepared from human blood samples, and CD16+ cells are sorted from the monocytes as reference cells in single-cell sequencing. This group is named reference standard cells (RSC cells for short). Different numbers of RSC cells are added to the samples to be sequenced for single-cell sequencing to test the role of the reference cells in single-cell sequencing. The specific steps include:

[0060] Step C1: Obtain a blood sample and store it in an EDTA anticoagulant tube at 4°C.

[0061] Step C2: Filter the blood sample through a 40 μm cell sieve to observe whether there is coagulation and remove corresponding impurities.

[0062] Step C3: Add an equal amount of DPBS to dilute the blood sample by half.

[0063] Step C4: Add 4 ml of Ficoll to the bottom of a 15 ml centrifuge tube, add 4 ml of blood diluted one-fold with DPBS and spread it evenly in the 15 ml centrifuge tube pre-added with Ficoll, so that the blood and Ficoll present a layered structure.

[0064] Step C5: After tiling, centrifuge at 1000×g, 20°C, for 20 min to produce layers and collect mononuclear cells.

[0065] Step C6: After centrifugation, four layers will appear. The first layer is the plasma layer, the second layer is the mononuclear cell layer, the third layer is the mixed layer of DPBS and Ficoll solution, and the last layer is the blood cell and neutrophil layer. Use a 200μl pipette to insert into the middle of the liquid to absorb the middle mononuclear cell layer.

[0066] Step C7: After the aspiration is completed, add 4 ml of DMEM + 0.04% BSA culture medium and centrifuge at 500×g, 5 min, 4°C to enrich mononuclear cells.

[0067] Step C8: After centrifugation, discard the supernatant and add 7 ml of DMEM + 0.04% BSA culture medium. Centrifuge at 500×g, 5 min, 4°C, and wash the mononuclear cells by low-speed centrifugation.

[0068] Step C9: Enrich CD16+ monocytes using CD16 positive magnetic beads.

[0069] Step C10: After low-speed centrifugation, discard the supernatant, add 1 ml of DMEM + 0.04% BSA culture medium to resuspend the cell pellet and centrifuge to obtain the final CD16+ monocyte suspension, named: RSC cells.

[0070] Step C11: Observe and count the CD16+ monocyte suspension, and calculate the cell viability.

[0071]

[0072]

[0073] Step C12: The final cells obtained are high-quality monocyte samples. The errors related to the previous experiments are eliminated. The single-cell suspension is loaded onto the machine using the Mozo platform. The same single-cell sample is used to add 0, 200, 500, 1000, 3000, and 5000 RSC cells from the standard kit. A total of six groups are tested to determine the optimal added quality control concentration of the standard kit cells.

[0074] Example 2: A kit method for single-cell sequencing quality control standards 1

[0075] 1. Preparation of Monocytes

[0076] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0077] 2. Preparation of standard cells

[0078] The RSC cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely thaw. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4 Concentration for standby use;

[0079] 3. Get on the machine

[0080] Configure the mix of Mozhuo machine to add 3×10 4 After that, 5×10 2 The remaining RSC standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0081] 4. Sequencing Analysis

[0082] The illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of RCS.

[0083] 5. Test results

[0084] The final RCS data is as follows Figure 4 The Estimated Number of Cells is 430, the Median Genes per Cell is 1535, the Mean Reads per Cell is 13509, and the Median UMI Counts per Cell is 3638.

[0085] Example 3: A kit method for single-cell sequencing quality control standards 2

[0086] 1. Preparation of Monocytes

[0087] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0088] 2. Preparation of standard cells

[0089] The RSC cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely thaw. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4

[0090] Concentration for standby use;

[0091] 3. Get on the machine

[0092] Configure the mix of Mozhuo machine to add 3×10 4 After high-viability monocytes were injected into the 3 The remaining standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0093] 4. Sequencing Analysis

[0094] The illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of RCS.

[0095] 5. Test results

[0096] The final RCS data is as follows Figure 5The Estimated Number of Cells is 896, the Median Genes per Cell is 1505, the Mean Reads per Cell is 13219, and the Median UMI Counts per Cell is 3503.

[0097] Example 4: A kit method for single cell sequencing quality control standards 3

[0098] 1. Preparation of Monocytes

[0099] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0100] 2. Preparation of standard cells

[0101] The RSC cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely thaw. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4

[0102] Concentration for standby use;

[0103] 3. Get on the machine

[0104] Configure the mix of Mozhuo machine to add 3×10 4 After high-viability monocytes were injected into the 3 The remaining RSC standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0105] 4. Sequencing Analysis

[0106] The illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of RCS.

[0107] 5. Test results

[0108] The final data is as follows Figure 6 The estimated number of cells is 1641, the median genes per cell is 1557, the mean reads per cell is 14285, and the median UMI counts per cell is 3693.

[0109] Example 5: A kit method for single cell sequencing quality control standards 4

[0110] 1. Preparation of Monocytes

[0111] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0112] 2. Preparation of standard cells

[0113] The RSC cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely thaw. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4

[0114] Concentration for standby use;

[0115] 3. Get on the machine

[0116] Configure the mix of Mozhuo machine to add 3×10 4 After high-viability monocytes were injected into the 3 The remaining RSC standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0117] 4. Sequencing Analysis

[0118] The illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of RCS.

[0119] 5. Test results

[0120] The final RCS data is as follows Figure 7 The Estimated Number of Cells is 2657, the Median Genes per Cell is 1535, the Mean Reads per Cell is 14143, and the Median UMI Counts per Cell is 3609.

[0121] Example 6: A kit method for single cell sequencing quality control standards 5

[0122] 1. Preparation of Monocytes

[0123] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0124] 2. Preparation of standard cells

[0125] The RSC cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely thaw. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4

[0126] Concentration for standby use;

[0127] 3. Get on the machine

[0128] Configure the mix of Mozhuo machine to add 3×10 4 After that, 5×10 3 The remaining RSC standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0129] 4. Sequencing Analysis

[0130] The Illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of RCS.

[0131] 5. Test results

[0132] The final RCS data is as follows Figure 8 The Estimated Number of Cells is 3364, the Median Genes per Cell is 1567, the Mean Reads per Cell is 14753, and the Median UMI Counts per Cell is 3705.

[0133] Example 7: A kit method for single-cell sequencing quality control standards 5

[0134] 1. Preparation of Monocytes

[0135] The Ficoll method was used to separate PBMC to obtain low-viability monocytes for subsequent sequencing experiments;

[0136] 2. Preparation of standard cells

[0137] The cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely melt. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4 Concentration for standby use;

[0138] 3. Get on the machine

[0139] Configure the mix of Mozhuo machine to add 3×10 4After low-viability monocytes were injected into the 3 The remaining standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0140] 4. Sequencing Analysis

[0141] The illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of RCS.

[0142] 5. Test results

[0143] The final RCS data is as follows Fig. 9 The Estimated Number of Cells is 986, the Median Genes per Cell is 1487, the Mean Reads per Cell is 24945, and the Median UMI Counts per Cell is 3649.

[0144] Comparative Example 1: A kit method for single-cell sequencing quality control standards 1

[0145] 1. Preparation of Monocytes

[0146] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0147] 2. Preparation of standard cells

[0148] The cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely melt. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4 Concentration for standby use;

[0149] 3. Get on the machine

[0150] Configure the mix of Mozhuo machine to add 3×10 4 After adding 10 high-viability monocytes, add 0 RSC standard cells into it, and use DEPC water to make up the remaining system to the final cell system on the machine;

[0151] 4. Sequencing Analysis

[0152] The Illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of monocytes.

[0153] 5. Test results

[0154] The final data of monocytes are as follows Figure 3 The Estimated Number of Cells is 20532, the MedianGenes per Cell is 2409, the Mean Reads per Cell is 18391, and the Median UMI Counts per Cell is 5176.

[0155] Comparative Example 2: A kit method 1 for single-cell sequencing quality control standards

[0156] 1. Preparation of Monocytes

[0157] The Ficoll method was used to separate PBMC to obtain high-viability monocytes for subsequent sequencing experiments.

[0158] 2. Preparation of standard cells

[0159] The cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely melt. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4 Concentration for standby use;

[0160] 3. Get on the machine

[0161] Configure the mix of Mozhuo machine to add 3×10 4 After high-viability monocytes were injected into the 3 The remaining RSC standard cell system is supplemented with DEPC water to the final cell system for the machine;

[0162] 4. Sequencing Analysis

[0163] The Illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of monocytes.

[0164] 5. Test results

[0165] The final data of monocytes are as follows Fig.10 The Estimated Number of Cells is 18953, the MedianGenes per Cell is 2128, the Mean Reads per Cell is 24917, and the Median UMI Counts per Cell is 4612.

[0166] Comparative Example 3: A kit method 1 for single-cell sequencing quality control standards

[0167] 1. Preparation of Monocytes

[0168] The Ficoll method was used to separate PBMC to obtain low-viability monocytes for subsequent sequencing experiments.

[0169] 2. Preparation of standard cells

[0170] The cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely melt. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 10 4 Concentration for standby use;

[0171] 3. Get on the machine

[0172] Configure the mix of Mozhuo machine to add 3×10 4 After adding 10 low-viability monocytes, add 0 RSC standard cells into it and use DEPC water to make up the remaining system to the final cell system on the machine;

[0173] 4. Sequencing Analysis

[0174] The Illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of monocytes.

[0175] 5. Test results

[0176] The final data of monocytes are as follows Fig.11 The Estimated Number of Cells is 21,998, the MedianGenes per Cell is 974, the Mean Reads per Cell is 24,223, and the Median UMI Counts per Cell is 1,715.

[0177] Comparative Example 4: A kit method 1 for single-cell sequencing quality control standards

[0178] 1. Preparation of Monocytes

[0179] The Ficoll method was used to separate PBMC to obtain low-viability monocytes for subsequent sequencing experiments.

[0180] 2. Preparation of standard cells

[0181] The cells in the standard kit were placed in a 37°C constant temperature water bath and incubated for 2 minutes to completely melt. After that, 4 ml of DMEM + 0.04% BSA medium was added and centrifuged for enrichment. After that, an appropriate amount of DMEM + 0.04% BSA was added to dilute the cells to 5 × 104 Concentration for standby use;

[0182] 3. Get on the machine

[0183] Configure the mix of Mozhuo machine to add 3×10 4 After low-viability monocytes were injected into the 3 RSC standard cells, and the remaining system is supplemented with DEPC water to the final cell system on the machine;

[0184] 4. Sequencing Analysis

[0185] The Illumina platform was used to measure 100G data volume for subsequent analysis. After obtaining the original data, the data was split to obtain the final cellranger map of monocytes.

[0186] 5. Test results

[0187] The final data of monocytes are as follows Fig.12 The Estimated Number of Cells is 17749, the MedianGenes per Cell is 820, the Mean Reads per Cell is 18194, and the Median UMI Counts per Cell is 1485.

[0188] The final results of the above examples and comparative examples are summarized in Table 1 below.

[0189] Table 1

[0190]

[0191] Analyzing the above table, we can get:

[0192] 1. From Examples 2-7, it can be seen that in the data of RSC added to monocytes, the number of RCS cells and the activity rate of the samples do not affect the value of Median Genes per Cell of the overall RCS. The Median Genes per Cell of RCS tends to be stable at around 1500. From the comparison between Examples 4 and 7, it can be seen that low-activity samples or high-activity samples will not affect the overall median gene value of RCS. From Examples 2-6, the median gene value of RSC does not produce a large deviation, which shows that the number of standard cells does not affect the median gene value of RSC. In summary, RSC can be used as a standard to measure the quality of samples;

[0193] 2. From Comparative Examples 1 and 2, it can be seen that for high-activity samples, the addition of RSC will not have much effect on the final data of the sample itself. The Median Genes per Cell of the mononuclear sample before and after the addition of RSC is still maintained at more than 2000, without a significant decrease; From Comparative Examples 3 and 4, it can be seen that for low-activity samples, the addition of RSC will not have much effect on the final data of the sample itself. The Median Genes per Cell of the mononuclear sample before and after the addition of RSC is still maintained below 1000, without a significant increase;

[0194] 3. In summary, 1 and 2 above show that the addition of RSC will not cause the Median Genes per Cell of the sample itself to increase or decrease. The addition of RSC can be used to determine the quality of the sample itself. When the final data quality of the sample shows that Median Genes per Cell is low, if the Median Genes per Cell of RSC is maintained at a normal level of around 1500, but the Median Genes per Cell of the sample itself is below 1000, it can be judged that the quality of the sample itself is poor. If both the Median Genes per Cell of RSC and the Median Genes per Cell of the sample itself show a significant decrease, it can be judged that there is a problem with the quality of the kit, resulting in an abnormality in the overall Median Genes per Cell.

[0195] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A quality control kit for single-cell sequencing, characterized in that: The kit comprises a plurality of individually packaged CD16+ human peripheral blood mononuclear cell suspensions, wherein the number of cells contained in the CD16+ human peripheral blood mononuclear cell suspension is known.

2. The kit according to claim 1, characterized in that The kit includes 5 separately packaged CD16+ human peripheral blood mononuclear cell suspensions with known cell numbers, and the cell number of each CD16+ human peripheral blood mononuclear cell suspension is different.

3. The kit according to claim 1, characterized in that The kit comprises five individually packaged CD16+ human peripheral blood mononuclear cell suspensions, wherein the numbers of CD16+ human peripheral blood mononuclear cells contained in the five CD16+ human peripheral blood mononuclear cell suspensions are 200, 500, 1000, 3000 and 5000, respectively.

4. The kit according to claim 3, characterized in that The composition of the kit is as follows:

5. Use of CD16+ human peripheral blood mononuclear cells as quality control for single-cell sequencing.

6. A method for preparing CD16+ human peripheral blood mononuclear cells, characterized in that: The method comprises the steps of: S1: Obtain human peripheral blood mononuclear cells; S2: CD16+ monocytes are obtained by sorting.

7. The method according to claim 6, characterized in that The method of obtaining human peripheral blood mononuclear cells comprises the steps of: S11: Obtain a blood sample and filter out impurities therein through a cell sieve; S12: Dilute the filtered blood sample with a diluent, and then add an appropriate amount of Ficoll; S13: Centrifugation to layer the blood sample with Ficoll and collect mononuclear cells; S14: Cultivate the obtained monocytes in culture medium to obtain.

8. The method according to claim 7, characterized in that The monocytes obtained by culturing in a culture medium include the steps of: culturing the monocytes obtained in 4 ml of DMEM culture medium containing 0.04% BSA, and then centrifuging to enrich the monocytes. Preferably, the centrifugation to enrich the monocytes is performed at 4° C. for 5 minutes, and the centrifuge speed is 500×g.

9. A single-cell sequencing method, characterized in that: The method comprises the steps of: adding CD16+ human peripheral blood mononuclear cell quality control product to the sample to be sequenced for detection.

10. The method according to claim 9, characterized in that The CD16+ human peripheral blood mononuclear cell quality control product contains CD16+ human peripheral blood mononuclear cells prepared by the method according to any one of claims 6 to 8.

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