GSM8880593: Granulosa, rep2; Mus musculus; OTHER
Following dissection, gonads were separated from the mesonephros using a G25 needle and dissociated into single cells by culture in 0.045% trypsin- EDTA (Thermo, cat. 25300062) along with 0.25% collagenase (Wortington, cat. LS004176) in a total volume of 500 µl  for 8 min at 37°C in a 24-well plate. 200 µl of DPBS (without Calcium and Magnesium) containing 3% Bovine serum albumin (BSA) (Sigma-Aldrich, cat. A3311) were added to quench the trypsin action and immediately aspirated to remove the trypsin. Gonads were then resuspended in 300µl of DPBS containing 3% BSA and mechanically dissociated by gentle pipetting. Cells were filtered through a 30µm strainer (Miltenyi Biotec, cat. 130-041-407) and collected into a Polypropylene FACS tube (BD Falcon, cat. 352063). The well and filter are washed with additional 200µl of DPBS containing 3% BSA. Cells were sorted using the BD FACS Aria III cell sorter with an 85-micron nozzle. Texas Red filter (excitation 561 nm, emission 610 nm) was used to detect mCherry and FITC (excitation 495 nm, emission 519 nm) filters to detect GFP. For ATAC sequencing, positive cells were sorted into Eppendorf tubes containing 150µl of DPBS + 3% BSA. For RNA sequencing positive cells were collected into 150µl QIAzol lysis reagent (Qiagen, 79306). FACS raw data were analysed using FlowJo v10 for visualization. Capture Hi-C libraries (3 libraries of E13.5 Sox9IRES-GFP Sertoli cells and 2 libraries of E13.5 Enh8-mCherry pre-granulosa cells) were generated from 100,000 sorted cells, crosslinked as previously described (92, 93). Following lysis, the nuclei were permeabilized and digested with DpnII (NEB) overnight. The restriction overhangs were filled in using a biotinylated dATP (Jena Bioscience) and ligation was performed for 4 hours at 16°C (T4 DNA ligase; Life Technologies). The crosslinks were reversed using proteinase K and overnight incubation at 65°C, followed by purification with SPRI beads (AMPure XP; Beckman Coulter). Short fragments up to 1000 bp were produced via tagmentation and the biotinylated restriction junctions were then pulled down using MyOne C1 streptavidin beads (Life Technologies). PCR amplification (5 cycles) was performed on the libraries directly bound to the C-1 beads and the libraries were purified using SPRI beads as before. Promoter Capture was performed using the mouse custom-designed Agilent SureSelect system, following the manufacturer's protocol, followed by 7 PCR cycles. The libraries were sequenced using 150 bp paired-end sequencing on an Illumina NovaSeq (Novogene UK) with a sequencing depth of 1bln paired reads for each biological replicate.