GSM6468360: human1, cd45+, scRNAseq; Homo sapiens; RNA-Seq
Resected tumor sample were examined by a genitourinary pathologist who confirmed the pathological staging and tumor content. Human tumors were then resected and dissociated to the single cell level in a solution of RPMI-10% FBS (Gibco) + 1% type 1 collagenase (Gibco). The dissociate is subject to mechanical dissociation through a 16G needle and syringe (5-10 passes) and then a 21.5G needle (5-10 passes). Single cell dissociates were then sorted for live CD45 positive cells and sorted using a BD Aria after cell staining using a BV510-CD45 antibody (Biolegend) and DAPI stain. Single cell suspensions of all sorted samples were then resuspended in BSA (0.2 % in PBS) at a concentration of 2x10^6 cells/ml followed by barcoding with a 10x Chromium Controller (10x Genomics). To prepare tissues for scRNA-seq, human tissues were enzymatically and mechanically digested to the single-cell level, flow cytometry sorted for CD45-positive and CD45-negative cells followed by processing using the 10x Chromium platform. Sequencing was done on a Nextseq 500 platform and analyzed using Cell Ranger V2.