GSM5289095: RB_128_Rep_2; Homo sapiens; RNA-Seq
Tumor and xenograft samples were dissociated using an enzymatic tumor dissociation. The tumor was added to 10 mL of RPMI with 600 μl of trypsin (10 mg/ml, Sigma Cat#T9935) and incubated at 37C for 10 minutes. 600μL of each Soybean Trypsin Inhibitor (10 mg/ml) DNase I (2mg/mL) and magnesium chloride (1 M) were added and the tumor suspension was filtered through a 40 uM cell strainer and centrifuged at 500 rcf. The pellet was resuspended in 5 ml of red blood cell lysis solution (5 Prime Cat#2301310) and incubated at RT for 10 minutes. 5mL PBS -/- with 10% FBS was added and the cell suspension centrifuged at 500 rcf for 5 minutes. Supernatant was discarded and cell pellet was resuspended in RPMI. The cell suspension was then layered on top of a BSA cushion (4% BSA in REM) and centrifuged at 500g for 10 minutes. The supernatant was removed and the cells were resuspended at ~1000 cells/uL and counted. 10x genomics 3' scRNA-seq v3.1 was used to construct the single cell library