title: GSM7830772: Fetal_rete_ovarii_scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: 28 ROs at E16.5 and E17.5 were collected as described above, pooled and incubated for 12 min in 450ul Trypsin 0.05% with 50ul 2.5% Collagenase Type IV at 37ºC. 500ul of chilled PBS 0.3% BAS were then added for mechanical disaggregation by pipetting gently up and down for about 2 min. Some tissue chunks were still present, thus the sample was incubated for another 6 min in 100ul Trypsin 0.05% with 50ul 2.5% Collagenase Type IV at 37ºC. The sample was then pelleted by gentle centrifugation (5 min 500 x g 4ºC) and resuspended in 500ul Red Blood Cell lysis buffer (eBioscience, cat #00-4333-57) and incubated for 3 min at room temperature. Cells were pelleted by gentle centrifugation and resuspended in 120ul PBS 0.3% BSA before passing through a 0.32um cell strainer into a clean 1.5mL tube. To capture, label, and generate cDNA libraries of individual cells and nuclei, the 10X genomics Chromium Single Cell 3' Library and Gel Bead Kit v3 following the 10X Genomics User Guide was used. 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.