An iPSC residue detection method based on single-cell sequencing data analysis
A single-cell sequencing and data technology, applied in the field of biomedicine, can solve the problem of high false negative rate, and achieve the effect of high accuracy, high detection efficiency and high sensitivity
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Embodiment 1
[0098] Example 1 Detection of residual iPSC in endothelial progenitor cells (EPC)
[0099] 1. Experimental materials
[0100] E8 complete medium, stem pro 34 basal medium, DMEM / F12 medium, TrypLE, BMP4, HumanRecombinant VEGF165 (VEGFA), Forskolin, Human Recombinant Activin A were purchased from Thermofisher; Y-27632, CHIR99021, SB431542 were purchased from Sigma company; matrigel, Fibronectin purchased from Corning.
[0101] 2. The process of iPSC differentiation into EPC cells
[0102] (1) According to the iPSC passaging procedure, after the cells were centrifuged normally, remove the supernatant, add an appropriate amount of E8 complete medium containing 10 μM Y-27632 pre-warmed at 37°C, and gently pipette to resuspend the cell pellet, and then resuspend the cells. Cell fluid count and viability detection;
[0103] (2) From 37℃, 5%CO 2 Take out 4 Matrigel-coated T75 culture flasks from the cell culture incubator, remove the liquid, and add 13 mL of E8 complete medium con...
Embodiment 2
[0144] Example 2 Detection of residual iPSC in cardiomyocytes
[0145] 1. Experimental materials
[0146] DMEM / F-12 medium, GlutaMAX TM Supplement, Penicilin-streptomycin (double antibody), BMP4, B27 were purchased from Thermofisher; RPMI-1640 medium was purchased from Hyclone; TESR-E8 was purchased from STEMCELL Technologies; Y-27632, CHIR99021, C59, IWR1, Thioglycerol and L-ascorbic acid were purchased from Sigma Company.
[0147] 2. The process of iPSC differentiation into cardiomyocytes
[0148] (1) When the iPSC cells are expanded to 75-85% degree of polymerization, the passage begins. Take the T25 culture dish as an example, remove the old medium, wash twice with room temperature PBS, and then add 3 mL of EDTA preheated at 37°C. Working solution, placed at 37°C, 5% CO 2 In the cell incubator for 5 min, observe the gap between single cells under the microscope, discard the EDTA, add 3 mL of TeSR-E8 complete medium to terminate the digestion, transfer to a 15 mL centr...
Embodiment 3
[0184] Example 3 Single-cell sequencing of islet cells
[0185] 1. Islet cell single-cell sequencing data analysis process
[0186] (1) Use cellranger-5.0.0 to analyze single-cell transcriptome rawdata data, use the cellranger count tool, the reference genome version is GRCh38-2020-A, and analyze the EPC single-cell transcriptome expression matrix results:
[0187] cellranger count--id=EPC--fastqs=rawdata_dir--sample=EPC--localcores=8--localmem=64--transcriptome=refdata-gex-GRCh38-2020-A;
[0188] (2) The Seurat software package can analyze single-cell data. First, use the R function Read10X to read the EPC single-cell transcriptome expression matrix to obtain a sparse matrix, create a Seurat object, and set conditions to screen cells:
[0189] pbmc.data<-Read10X(data.dir=data_dir)
[0190] pbmc1<-CreateSeuratObject(counts=pbmc.data,project=project_name,min.cells=QC_min_cells,min.features=QC_min_features)
[0191] Among them: data_dir is the directory where the single-cell ...
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