title: GSM8478325: SCC0002_2_Aguilar Multiseq; Mus musculus; OTHER
library_construction_protocol: Prostates were minced into small pieces and digested in a two step protocol: with  5 mg mL-1 for 1 h at 37°C on a shaking platform; and then with TrypLE Express Enzyme for 15 min at 37°C  and mechanically disrupted every 5 min. Organoids and organoid-derived monolayers were harvested with TrypLE Express Enzyme for 15 min at 37°C  and mechanically disrupted every 5 min.  Cells were labelled with CMO tags (ex vivo units: CMO 1, 2; 3D organoids Ctrl: CMO 3; 3D organoids DHT: CMO 4; 2D organoids Ctrl : CMO 5, 6; 2D organoids DHT: CMO 7, 8). After labeling, cells were washed pooled in equal proportions in two different pools (ex vivo and 3D: 1300 cells/uL, 80% viability; 2D: 902 cells/uL; 86% viability). The pooled cells were subsequently loaded onto a Chromium Single Cell Controller (10x Genomics) to generate Gel Bead-In-Emulsions (GEMs). Post-GEM-RT cleanup was performed using Dynabeads MyOne Silane Beads (37002D, Thermo Fisher Scientific). cDNA was amplified by PCR and then fragmented, end-repaired, and A-tailed. Adapters were ligated, and the resulting constructs were enriched by PCR to generate the final libraries. Libraries were quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and their size distribution was assessed using an Agilent 2100 Bioanalyzer. Libraries were sequenced using Illumina NovaSeq6000 using a S2 flow cell with 100 cycles. Single cells were encapsulated into droplets with the Chromium™ Controller (10x Genomics). Transcripts captured in all cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 12 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3' reagents kit v3.1 (10x Genomics) including cell multiplexing via CellPlex kit Set A  following the detailed protocol provided by the manufacturer (CG000390 rev B, 000391 Rev B). 10x Gemoics library construction kit Cell multiplexing via 10x Genomics CellPlex Prostates were minced into small pieces and digested in a two step protocol: with  5 mg mL-1 for 1 h at 37°C on a shaking platform; and then with TrypLE Express Enzyme for 15 min at 37°C  and mechanically disrupted every 5 min. Organoids and organoid-derived monolayers were harvested with TrypLE Express Enzyme for 15 min at 37°C  and mechanically disrupted every 5 min.  Cells were labelled with CMO tags (ex vivo units: CMO 1, 2; 3D organoids Ctrl: CMO 3; 3D organoids DHT: CMO 4; 2D organoids Ctrl : CMO 5, 6; 2D organoids DHT: CMO 7, 8). After labeling, cells were washed pooled in equal proportions in two different pools (ex vivo and 3D: 1300 cells/uL, 80% viability; 2D: 902 cells/uL; 86% viability). The pooled cells were subsequently loaded onto a Chromium Single Cell Controller (10x Genomics) to generate Gel Bead-In-Emulsions (GEMs). Post-GEM-RT cleanup was performed using Dynabeads MyOne Silane Beads (37002D, Thermo Fisher Scientific). cDNA was amplified by PCR and then fragmented, end-repaired, and A-tailed. Adapters were ligated, and the resulting constructs were enriched by PCR to generate the final libraries. Libraries were quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and their size distribution was assessed using an Agilent 2100 Bioanalyzer. Libraries were sequenced using Illumina NovaSeq6000 using a S2 flow cell with 100 cycles.