GSM8124730: AV3_V2; Mus musculus; RNA-Seq
Single-cell suspensions from healthy colon or dysplastic lesions were processed using a modified version of a previously published protocol40. Tissue samples were rinsed in 30ml of ice-cold PBS (ThermoFisher 10010-049), chopped to small pieces and washed twice in 25 ml PBS, 5mM EDTA (ThermoFisher AM9261), 1%FBS (ThermoFisher 10082-147). To prime tissue for enzymatic digestion, samples were incubated for 10 minutes at 37°C, placed on ice for 10 minutes before shaking vigorously 15 times followed by supernatant removal. Tissues were placed into a large volume of ice-cold PBS to rinse prior to transferring to 5ml of enzymatic digestion mix (Base: RPMI1640, 10 mM HEPES (ThermoFisher 15630-080), 2% FBS), freshly supplemented immediately before use with 100 mg/mL of Liberase TM (Roche 5401127001) and 50 mg/mL of DNase I (Roche 10104159001), and incubated at 37°C with 120 rpm rotation for 30 minutes. After 30 minutes, enzymatic dissociation was quenched by addition of 1ml of 100% FBS and 10mM EDTA. Samples were then filtered through a 40 mM cell strainer into a new 50 mL conical tube and rinsed with PBS to 30 mL total volume. Tubes were spun down at 400 g for 7 minutes, at 4°C. Resulting cell pellets were resuspended in 1ml PBS, placed on ice and counted. 10XV2, following the manufacturer protocol for the v2 kit (10X Genomics, Pleasanton, CA).