GSM7091763: DLVP-Castrated, replicate 1, scRNA; Mus musculus; RNA-Seq
Single cell tissue dissociations were performed as previously described (Lukacs et al., 2010). Briefly, in dissecting medium (DMEM (#10-027-CV, Corning) with 10% (vol/vol) FBS (#10437028, ThermoFisher), 1X glutaMAX (#35050061, ThermoFisher), and 1X penicillin-streptomycin (#15070063, ThermoFisher)) prostates were dissected away from the GU tract into individual lobes from 4-6 independent mice (N=4-6). Dorsal, lateral, and ventral prostatic lobes were pooled together, and anterior lobes were processed separately. Pooled prostatic tissue was minced and transferred to digestion solution containing 1mg/ml collagenase (#17100017, ThermoFisher Scientific). Tissue was digested at 37 °C for 2 hours with constant inversion. After digestion, tissue was spun down, resuspended in Trypsin-EDTA (#25300120, ThermoFisher), and digested for an additional 5 minutes at 37 °C. Trypsin digestions was inhibited by addition of dissecting medium supplemented with 500 U DNase I (#10104159001, Millipore-Sigma). Digested tissue was next passed through 18-G and then 20-G syringes to facilitate dissociation. Dissociated tissue was finally passed through a mesh filter with a 40 mm pore size to isolate single cells. All solutions used for tissue digestion/dissociation contained 10 mM Y-27632 dihydrochloride (#1254, Tocris) to prevent anoikis. Cell viability was greater than 80% for all downstream assays. scRNA-Seq for dissociated mouse tissues was performed using 10X genomics Chromium Single Cell 3' Library & Gel bead Kit V2 (ADLVP study) or the 10X genomics Chromium Single Cell 3' Library & Gel bead Kit V3 (AP2 study) according to the manufacturer's protocol. Briefly, approximately 17,000 viable cells were loaded into each channel of the Chromium Next GEM Chip G to capture 10,000 Gel Beads-in-emulsion (GEMs) per channel. After GEM capture, gel beads are dissolved, and cells are lysed into the cDNA master mix containing reverse transcription (RT) reagents. Post RT incubation, GEMs were broken, and first strand barcoded-cDNA was purified with magnetic beads, followed by 12-cycles of PCR-amplification. PCR-amplified barcoded-cDNA was then fragmented and purified with SPRI beads to obtain an average fragment size of 600 bp. Next, cDNA libraries were ligated to sequencing adapters followed by indexing PCR. Resulting libraries were sequenced on the Illumina NovaSeq 6000. Cell-ranger v2.1.0 (10X genomics) was used to demultiplex all mouse runs to FASTQ files, align reads to the mm10 mouse transcriptome and extract cell and UMI barcodes. The pipeline output is a cell by gene count matrix, which records the number of UMIs for each gene associated with a particular cell barcode.