Mus musculus
Protocols: GEMM mice prostates were dissected in cold PBS1X containing RNAi (ratio 1:1000) SUPERASE RNase INHIBITOR in cold small petri dish and disrupted mechanically in small pieces with blade. The minced tissues were moved to 3 ml of digestion medium: RPMI-1640; 10% FBS; Penn/Strept 1%; HEPES 25 mM; Collagenase D, 1 mg/ml; DNase I Solution (2500 U/mL). Tissues were incubated for 50 min at 37°C on orbital shaker. After, digestion was blocked by adding 3 ml of stopping buffer (RPMI-1640 with 10% FBS and 1% Penn/Strept). Samples were homogenized with a 1 ml pipette and filtered through a 100 µm nylon mesh and rinsed with 4 ml of RPMI-1640 medium. Cell suspension was let to rest for 4 min on ice and then filtered through 40 µm nylon mesh strainer. After centrifugation at 1500 rpm for 5 minutes at 4°C, cells were resuspended in 200 µL of stopping buffer and vortexed in FACS sorting tubes (Falcon™ Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5mL). Cell suspension was incubated for 5 minutes with 7-AAD (7-Aminoactinomycin D) before sorting performed with FACS ARIA (BD). At the end of sorting, viable sorted cells were collected in low binding 1.5mL eppendorf tubes pre-coated with BSA 3% and 10 µl RNAi and 15 µl of RPMI-1640 with FBS 10%. Not applicable Chromium Next GEM Single Cell 3' guide was followed.