GSM8257393: MRC5; Mus musculus; RNA-Seq
To prepare prostate tumor cells suspension, prostates were digested with digestion buffer (5 mg/ml Collagenase II  in Advanced DMEM-F12 with Y-27632) for 20 min at 37°C with agitation, filtered through 70- and 40-μm-mesh cell strainers, washed with Advanced DMEM-F12 plus Glutamax 1x, Hepes 10 mM, 10 uM Y-27632, and Pen/Strep 1x (ADF 4+). Cells were further digested eith TryplE during 20 min at 37°C, washed in ADF 4+, and resuspended with Advanced DMEM-F12 containing 10 mM HEPES, 1X Glutamax, 10 mM Y-27632, Pen/Strep 1x and 5% FBS. Dead cells were removed by EasySep Dead Cell Removal (Annexin V) kit (STEMCELL technologies). scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics),  (Single Cell 3′ library and Gel Bead Kit v.2) as described in the manufacturer's protocol (10x User Guide, Revision B). On average, approximately 20,000 cells were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer's protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq6000 instrument using a S1 flowcell.