GSM4314857: CD8-scc1_GE; Homo sapiens; OTHER
Patient tumors were obtained on the day of surgery and washed in cold 1X PBS. Any fat was removed from tumor samples using a scalpel. Then, tumors were then cut into small pieces using a razor and resuspended in 10 mL of RPMI [Gibco 11875-093] supplemented with 5% human serum [Sigma H4522-100], 5 mg gentamicin [Sigma H0887], 5 mg/mL collagenase II [Sigma C2674] and 10 U DNase I [Sigma 00453869] for 10 minutes at 37Â°C. After incubation, the suspension was vortexed 1X for 30 sec followed by pipetting sequentially through 25-mL, 10-mL, and 5-mL pipettes for 1 min each. Next, the cell suspension was filtered through a 70-_m filter [Fisher 22363548] and spun at 450 g for 10 minutes. After spinning, the supernatant was removed, and ACK red blood cell lysis was performed according to the manufacturerâ€™s protocol as needed [Gibco a10492-01]. Cells were then stained as described below prior to cell sorting and FACS analysis. The sorted cellular suspensions were loaded on a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). Approximately 10-12e3 cells were loaded per channel. Single-cell RNA-Seq libraries were prepared using the following Single Cell 5â€™ Reagent Kits: Chromiumâ„¢ Single Cell 5â€™ Library & Gel Bead Kit, PN-1000006 and Chromium Single Cell VDJ enrichment kit for Human T Cells (PN-100000). Libraries were run on a NovaSeq 6000 SP or S1 flow cell (depending on sample numbers) for both 5â€™ transcriptome data and TCR clonotype determination. scRNA-seq and scVDJ-seq
GEO Accession
GSM4314857