Illumina NovaSeq 6000 sequencing; A comprehensive single-cell map of mouse gonadal development at stages E10.5, E11.5 and E12.5 - 10x scRNAseq
A comprehensive single-cell map of mouse gonadal development at stages E10.5, E11.5 and E12.5 - 10x scRNAseq
Developing ovaries and testes were collected from E 10.5, 11.5, 12.5 and 13.5 mouse embryos carrying the Oct4ΔPE-GFP transgene. Developing gonads were dissected from the mesonephros and both organs were separately dissociated with 0.25% trypsin/EDTA into single cell suspensions as described for the humans. Males and female gonads and mesonephros were processed independently. For sectioning, tissues were fixed in 4% (w/v) formaldehyde solution for 2 hours and at 4 °C. Samples were washed with PBS and afterwards sequentially incubated with 10% and 20% (w/v) sucrose at 4°C. After, samples were embedded in an optimal cutting temperature compound (OCT) and subsequently flash frozen using a dry ice-isopentane slurry. All experimental procedures were carried out in agreement with the project licence PE596D1FE issued by the UK Home Office and carried out in a Home Office designated facility.  We used the previous protocol optimised for gonadal dissociation and available at protocols.io.  https://www.protocols.io/view/human-embryonic-gonad-dissociation-with-trypsin-ed-66fhhbn In short, tissues were cut into <1 mm segments before being transferred to a 15ml tube. Tissues were digested with Trypsin/EDTA 0.25% between 5-15 minutes at 37°C with intermittent shaking. Digested tissue was passed through a 100 µm filter, and cells were collected by centrifugation (500 x g for 5 minutes at 4°C). Cells were washed with flow buffer (PBS containing 5 % (v/v) FBS and 2 mM EDTA) prior to cell counting.       Cells were loaded according to the manufacturer's protocol for the Chromium Single Cell 5′ Kit v.2 (10X Genomics) to attain between 2,000 and 10,000 cells per reaction. Library preparation was carried out according to the manufacturer's protocol.