GSM4284244: P9_cSCC_scRNA; Homo sapiens; RNA-Seq
Fresh tumor specimens (patient or xenograft) and normal skin (patient only) were minced with a scalpel in serum-free DMEM (Gibco) with DNAse I (0.2 mg/mL, Worthington, Inc., Columbus, OH) to pieces <1 mm3 and placed in 5mL of 0.25% trypsin-EDTA (Gibco) at 37C for 30 minutes with trituration with a wide-bore pipet tip every 10 minutes. For normal skin, subcutaneous fat was removed with surgical scissors prior to mincing and trypsin-EDTA incubation. After 30 minutes, trypsin-EDTA was quenched with DMEM supplemented with 10% FBS and cells were filtered through a 70mm filter (BD Falcon). Dissociated cells were pelleted and washed once with cold serum-free DMEM with DNase I and were immediately cryopreserved in 90% SCC-13 media with 10% DMSO (Sigma-Aldrich). All steps other than trypsin incubation were completed at 4°C or on ice. For patients 3 and 8, samples were incubated in 0.25% trypsin-EDTA and type I collagenase 5mg/mL (Gibco) for 30 minutes (instead of trypsin-EDTA 0.25% alone for other patients), which led to recovery of higher numbers of T cells in normal skin samples from these patients. We did not observe major differences in downstream processing of cells recovered from collagenase dissociation. On the day of scRNA-seq droplet capture, cells were thawed in at least 5X dilution volume of SCC-13 media to wash off residual DMSO in cryopreservation media. Cells were pelleted and resuspended in cold phosphate buffered saline (PBS) (Gibco) with 2% FBS and 2mM EDTA (Invitrogen, Carlsbad, CA). Cells were stained with propidium iodide (Sigma-Aldrich) prior to sorting on a BD FACS Aria II. For patients 1 and 8, cells were stained with anti-CD45 antibody conjugated to V450 (clone HI30, 560367, BD Biosciences) to separate immune cells from non-immune cells. For patient 1, only CD45-negative cells were sequenced while for patient 8, both CD45+ and CD45- fractions were sequenced, neither of which affected downstream data processing. Sorted cells were pelleted and resuspended in PBS with 2% FBS and 2mM EDTA prior to scRNA-seq droplet capture 10X Genomics Chromium 3' v2 kit according to manufacturer's protocol
GEO Accession
GSM4284244