GSM9081840: TRMs from Omentum tumor, biological replicate 1,TCR; Homo sapiens; OTHER
Following tissue enzymatic dissociation of tissues, single-cell suspensions were subjected to antibody staining with anti-EPCAM, anti-CD56, anti-CD3 and anti-CD103. Cells were purified by fluorescence-activated cell sorting (FACS) on SONY MA400 cell sorter equipped with four lasers (405 nm, 488 nm, 561 nm and 638nm). A 100-μm nozzle running at 70 psi and 90 kHz was used as the setup for each sort session. TRM-like and circulating T cells were sorted from 4 different HGSOCs for scRNA-seq analysis. Single-cell RNA-sequencing was performed using the 10X Genomics Chromium. A single-cell suspension derived from dissociated tissue was sorted. The cell suspension was loaded onto the 10X Genomics Chromium Single Cell Controller at a concentration of 700~1000 cells per microliter in order to encapsulate between 5000 and 12,500 cells per sample. Briefly, the single cells, reagents, and 10x Genomics gel beads were encapsulated into individual nanoliter-sized Gelbeads in Emulsion (GEMs) and then reverse transcription of poly-adenylated mRNA was performed inside each droplet. The cDNA and VDJ-enriched libraries were completed in a single bulk reaction using the 10X Genomics Chromium Next GEM Single Cell v2 and V(D)J Reagent Kits. 25,000 or 5,000 sequencing reads per cell for scRNA-seq or VDJ libraries, respectively, were generated on the NovaSeq X Plus instrument. Demultiplexing, barcode processing, alignment, and gene counting were performed using the 10X Genomics CellRanger v7.0 software.