GSM6428970: PbCre_Pten/Rb 30 Wks Replicate 2, Intact (no castration or DHT); Mus musculus; RNA-Seq
Fresh benign and malignant GEMM and human biopsy 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 37 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 (Life technologies) with 10 μM Y–27632 dihydrochloride for 5–10 minutes at 37oC on a shaking platform until a single cell suspension was obtained. For human samples, 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. For GEMM tissue, all cells were subsequently FACS–sorted for singlets and viability using DAPI. Organoids were isolated from basement membrane extract digested to single-cell suspension by incubating with TrypLE (Life technologies) supplemented with Y-27632 (10 μM) for 10 min at 37 °C with shaking, followed by a 2-min incubation with EDTA (1 mM), Y-27632 (10 μM) in PBS0 and a second digestion with TrypLE (Life technologies) supplemented with Y-27632 for 5 min at 37 °C with shaking. Cells were filtered and resuspended in PBS0, Y-27632 (10 μM) and BSA (0.04%) before processing further for single-cell sequencing. Cells were checked for viability using 0.2% (w/v) Trypan Blue staining (Countess II) and all sequencing experiments were performed on samples with a minimum of 80% viable cells. Single-cell encapsulation and scRNA-seq library prep of FACS-sorted cell suspensions was performed on the Chromium instrument (10x Genomics) following the user manual (Reagent Kit 3' v2 for organoids and GEMM or v3 for HMP samples). Each sample loaded onto the cartridge contained approximately 5,000 - 10,000 cells at a final dilution of ~500 - 1000 cells/µl. Transcriptomes of encapsulated cells were barcoded during reverse transcription and the resulting cDNA was purified with DynaBeads, followed by amplification per the user manual. Next, PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to sequencing adapters and indexed by PCR. Indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on an Illumina HiSeq (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher) to a depth of >50 million reads per sample (>13,000 reads/cell).