Single-cell RNA-seq from human prostate cancer-adjacent benign prostatic hyperplasia
Single-cell RNA-seq from human prostate cancer-adjacent benign prostatic hyperplasia
A benign prostate sample was collected adjacent to the tumor (contralateral lobe) from a patient with prostate cancer at the University Hospital of Basurto at the time of robotic prostatectomy following Ugalde-Olano et al., Mehods, 2015. The biopsy was kept in MACS medium, tissue storage solution until dissociation. The tissue was washed with cold phosphate-buffered saline (PBS; GIBCO) and minced into fragments of less than 0.4 mm. It was digested in filter-sterilized TH-liberase solution (2.5mg/ml; Sigma Aldrich) in complete medium composed of DMEM (GIBCO), Pen/strep (GIBCO), 1M MgCl2 (Sigma Aldrich), 10 mg/ml DNAse I (Sigma Aldrich) and Y-27632 dihydrochloride (10 μM; AbMole) at 37°C shaking at 800r.p.m for 40 minutes. The tissue solution was manually disaggregated every 10 min during incubation and centrifuged at 2400xg for 5 min. Subsequently, it was digested with filtered TrypLE (Life Technologies) + Y-27632 dihydrochloride under constant pipetting for 2 min. TrypLE inactivation was performed by adding FBS (GIBCO) and centrifuged at 2400xg for 5 min. The cell pellet was resuspended in PBS and filtered through a 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 and then centrifuged at 2400xg for 5 minutes and resuspended all in 10% DMSO, 50% FBS and 40% RPMI. 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). 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).