GSM8110797: Erg+Pten- 4w; Mus musculus; ATAC-seq
For scATAC-seq, dorsal-lateral lobes of mouse prostates from indicated age were freshly dissociated (described above), and flow sorted for live single cells using DAPI. Fresh nuclei were isolated before processing for 10X Genomics Chromium single cell multiome ATAC + gene expression sequencing following manufacture's instructions. Mouse prostates were harvested and subsequently digested with collagenase type II (Gibco) for 2 hrs at 37 °C, followed by TrypLE (Gibco) digestion at 37 °C until a single-cell suspension was obtained. Digestions were supplemented with Y-27632 (10 μM) to inhibit anoikis and filtered to obtain single cell suspension. Murine prostate organoid culture was established and maintained under standard conditions as described previously122,123. Briefly, dissociated prostatic epithelial cells were embedded in 20-µl drops of Matrigel (Corning) and overlaid with mouse prostate organoid medium. Organoids were passaged by mechanical splitting by repetitively passing through a 200-µl pipette tip or by trypsinization using TryplE. For lineage marker analysis, organoids were trypsinized into single cells and seeded at 20,000-50,000 cells per well of a 12-well plate in the absence of EGF (counted as day 0). For EdU pulse-chase experiments, the organoid culture was incubated with EdU (10 µM, Theremo Fisher) for 2 hrs before harvesting (pulse) or wash-off and re-seeding (chase). To compare cell fate from reporter cells, basal and luminal cells were sorted based on single-positive reporter signals and seeded at equal cell number between sgNT and sgERG in the absence of EGF (counted as day 0). Basal cells were seeded at 20,000-50,000 cells per well of a 12-well plate; less luminal cells were seeded (5,000-10,000 per well) due to a low recovery from the sgERG group.