GSM8222916: Human Prostate Biopsy, HMP23B; Homo sapiens; RNA-Seq
Fresh benign and malignant tissue was mechanically cut using a scalpel into small pieces (~1–5 mm3). The tissue was then processed and dissociated in 5–10 mg/ml collagenase type II (Gibco) solution in adDMEM/F12+/+/+ with 10 µM Y–27632 dihydrochloride for 30 minutes to 2 hours on a 37oC shaking platform. This was followed by a 1 minute 0.5 M EDTA wash at room temperature, and subsequent digestion with TrypLE (Gibco) with 10 µM Y–27632 dihydrochloride for 5–10 minutes at 37oC on a shaking platform until a single cell suspension was obtained.  If more than 10% of doublets were present (visual inspection) or there was evidence for <80% viability (hemocytometer, using 0.2% Trypan Blue), then cells were FACS–sorted for singlets and viability using a DAPI. Please refer to methods of paper for further details. Dissociated cells were subjected to scRNA–seq using 10X genomics Chromium Single Cell 3' Library and Gel bead Kit (v3 for all human biospies, except v2 for HP95T as previously described PMID: 32355025) per manufacturer's protocol. ~3000 to 10,000 cells per sample was encapsulated and barcoded following the manual.  Sample viability varied between 72 and 95% (0.2% Trypan Blue).  The final sequencing libraries were double–size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina Nova–Seq platform (R1–26 cycles, i7–8 cycles, R2–70 cycles or higher).