GSM8653338: CD45+ sorted, Patient F (CG); frozen; Homo sapiens; RNA-Seq
Biopsy bites were immediately collected into DMEM media on ice, or put into freezing media (10% DMSO; 40% RPMI; 50% FBS) and frozen (for later CD45-enrichment). Fresh or frozen and thawed tissue was then manually dissociated with a sterile razor and transferred to pre-warmed 37°C digestion enzyme mix in RPMI (Miltenyi tumor dissociation kit, cat. 130-095-929). In a 37°C water bath, tissue digestion was agitated every 2 minutes, and pipetted up and down every 10 minutes for a total of 30 minutes. Digested tissue was filtered through a 70μM strainer, washed with RPMI containg 10% FBS, and the single-cell suspension was collected and processed for scRNAseq. Fresh samples were loaded immediately while frozen samples were sorted for CD45+ Live (Zombie-NIR-) cells. Library preparation was performed with the 10x Genomics Chromium Single Cell 5' v2 library kit (for both GEX and TCR libraries)