title: GSM8649351: XP; Mus musculus; RNA-Seq
library_construction_protocol: Fresh whole prostate tissues were dissected from genetically engineered mice and immediately processed for scRNA-seq analysis using the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1 (Dual Index). In brief, whole prostate tissues were minced and dissociated into single cell suspensions using the Worthington-Biochem papain dissociation kit (according to manufacturer instructions), followed by incubation in TryPLE + 10uM Y-27632 at 37C for 15min and passage through a 40um strainer (Flowmi). Cell count and viability were measured by trypan blue staining using Cell Countess II to ensure single cell suspension and viability >80%. If doublets/multiplets remained, the TrypLE dissociation steps were repeated. Cells were resuspended in RPMI+10%FBS for loading into the 10x Chip G for GEM generation, with a targeted cell recovery of 4000-5000 cells. For PIP-seq analysis of CD45+ immune cells, prostates were collected and dissociated into single cell suspensions, as described above, then FACS-sorted for live CD45+ cells. In brief, cells were stained with Zombie Aqua Live/Dead stain (Biolegend Zombie Aqua™ Fixable Viability Kit # 423101) for 30min at 4C for live/dead staining. And for CD45 staining, Fc block (TruStain FcX™ PLUS Antibody # 156603) was applied for 5-10min, followed by CD45+ APC stain (Biolegend APC anti-mouse CD45 Antibody # 147707) for 30min. Cells were washed and resuspended in 2%FBS + 25mM HEPES PBS for cell sorting into 20%FBS collection media. Sorted live CD45+ cells were then washed with PIP-seq cell resuspension buffer. 10X genomics libraries were prepared using 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1 (Dual Index) per manufacturer's protocol. Pip-seq libraries were prepared using the PIPseq™ T20 3ʹ Single Cell RNA Kit v3.0 per manufacturer's protocol.