title: GSM7830775: Adult_rete_ovarii_rep1 _bulkRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Pooled ROs (4-5 ROs for each replicate) were washed in RNase-free PBS-/- and incubated at 37°C in fresh 1X TrypLE (Thermo Fisher Scientific, catalog #12563029) for 20 minutes. TrypLE was then aspirated off, and samples were then resuspended in chilled RNase-free PBS with 3% bovine serum albumin (BSA) and pipetted gently up and down for about 2 min to disaggregate cells. Cells were pipetted through a 0.32μm cell strainer into FAC-sorting filter tubes (Corning Falcon cat # 352235)​​. Single-cell suspensions were then FAC-sorted at the Duke Flow Cytometry Shared Resource core on a B-C Astrios Sorter, based on presence or absence of GFP. Cells were sorted into PBS, pelleted, and resuspended in 10.5ul Clontech Lysis buffer (Takara Bio Inc. Cat # 635013). Sample libraries were prepared with SMART-Seq v4 Ultra Low Input RNA Kit (Takara Clontech Kit Cat# 63488) Embryonic day (E)16.5 fetuses were harvested from pregnant Tre-H2BGFPtg/tg dams crossed to a Pax8rtTAtg/+ male. Embryos positive for both alleles were identified by presence of green fluorescence in the urogenital epithelia. Male fetuses were discarded, and ovary/mesonephros/oviduct complexes from females were dissected in RNAse-free PBS-/-, with as little removal of surrounding tissue as possible to ensure retention and survival of RO cells. The GFP signal was then used to perform close dissection of the RO and surrounding ovarian capsule, and the oviduct was discarded. Ovaries and periovarian fat pads (in which the adult RO is embedded) were harvested from Pax8rtTA; Tre-H2BGFPtg/tg adult females. The RO was located within the fat pad by green fluorescence and closely dissected in RNase-free PBS-/-, with as much removal of surrounding tissue as possible to ensure enrichment in RO cells. To ensure capture of the IOR and CR, the dorso-lateral-most portion of the ovary was conserved, and the rest of the ovary was discarded. The samples were kept on ice until all samples were dissected. For snRNAseq analysis of the adult RO, samples were collected from 5 mice in early diestrus and 5 mice in estrus (total of 10 ROs in each group) and were snap frozen in an Ethanol/dry ice slush and stored at -80ºC until further processing.