|
The approach of single-cell dissociation was modified from previous published method34. The ovaries from C57 mice at PD1 were collected and cut into small pieces. Then the ovarian pieces (~5 ovaries/tube) were treated with 0.125% Trypsin (25200056, Thermofisher scientific) and 5 IU/ml RNase-free DNase I (EN0523, Thermofisher scientific) at 37 °C for total 15 minutes (5 mins × 3), then neutralized in DMEM-F12 medium (11320-033, Gibco) with 10% FBS (16000-044, Gibco). After digestion, the mixture was filtered through a 40-μm cell strainer (352340, BD Biosciences) to avoid any cell aggregations and then cells were centrifuged at 300 g for 5 min. Removed the supernatant and resuspended cell-precipitation with DMEM-F12 medium and 10% FBS. To separation somatic cells and oocytes as described protocol60, the ovarian single-cell mixture in 35 mm dish was incubated at 37°C, 5% CO2 for 8 hr. The supernatant contained oocytes was harvested and centrifuged at 300 g for 5 min. Removed the supernatant and resuspended cell-precipitation with DMEM-F12 medium (1% BSA).The libraries were constructed using the Chromium Single-Cell 3 Library Kit version 3.1 (10x Genomics, Dual Index). |
RNA-Seq |
TRANSCRIPTOMIC SINGLE CELL |
PolyA |
PAIRED
|
