title: GSM8289377: metastatic hormone naïve prostate cancer biopsy, patient 2; Homo sapiens; RNA-Seq
library_construction_protocol: Prostate tissue from prostate cancer patients. The biopsy was kept in MACS medium, tissue storage solution until dissociation. The tumor was washed with cold phosphate-buffered saline (PBS; GIBCO) and minced into fragments under 0.4 mm. The tissue was chemically dissociated in a sterilized filter with Liberase TH solution (2.5mg/ml; REF QZY-5401135001, Thomas Scientific) in complete media composed of Advanced DMEM/F-12 (REF 12-634-010, Fisher Scientific), Pen/strep (REF 15-140122, GIBCO), MgCl2 (5mM, REF M4880, Sigma Aldrich), DNAse I (200 μg/ml, REF 11284932001, Sigma Aldrich) and Y-27632 dihydrochloride (10 μM; AbMole) at 37ºC shaking at 800 r.p.m for 40 minutes. The tissue solution was manually disaggregated with a pipette every 10 min during the incubation followed by centrifugation at 2400xg for 5 min. Next, a second chemical dissociation was performed with TrypLE (REF 12605010, Thermo Scientific) supplemented with Y-27632 dihydrochloride under constant pipetting for 2 minutes. The inactivation of TrypLE was done by adding FBS (GIBCO) and centrifuged at 2400xg for 5 minutes. The cell pellet was resuspended in PBS, filtered through 40 μm filter and washed with PBS. The suspension was centrifuged at 2400xg for 5 minutes and resuspended in 200ul of PBS. Cells were counted on a Neubauer chamber and 20000 cells were then centrifuged at 2400xg for 5 minutes and resuspended in PBS for scRNAseq analysis according to manufacturers´ indications. Single cell library was prepared using “Chromium Next GEM Chip G Single Cell Kit”, 16 rxns PN-1000127, “Chromium Next GEM Single Cell 3ʹ Kit v3.1”, 4 rxns PN-1000269 and “Dual Index Kit TT Set A”, 96 rxns PN-1000215, following “Chromium Next GEM Single Cell 3 ' Reagent Kits v3.1 (Dual Index) User Guide” (Document number CG000315).