GSM5831854: 15wks_ivc; Macaca fascicularis; RNA-Seq
Fetal ovaries collected from monkey fetuses at 8-18 wpf were carefully dissected out in chilled PBS.  Fetal ovaries at 8-14 wpf were minced with scissors in 0.05% trypsin/EDTA/PBS solution (GIBCO, 15400-054), then incubated for 15 min at 37℃ with gentle pipetting every 5 min.  Dissociation medium [DMEM (GIBCO, 10313021) containing 10% FBS, 2mM L-glutamine (GIBCO, 25030081), 1X penicillin/streptomycin (GIBCO, 15070063), and 100μg/mL DNaseI (MERCK, DN25)] was added for further dissociation.  For fetal ovaries at 16 and 18 wpf, minced tissues were incubated in Accumax (Innovative Cell Technologies, AM105) for 40 min at 37℃ with gentle pipetting every 5 min.  Dissociation medium was added for further dissociation.  Cell suspensions were further treated with TryPLE Express (1X) (GIBCO, 12604021) for 5min at 37℃ to make the single-cell suspension. Dissociated cells from monkey fetal ovaries and cultured monkey rOvaries were stained with DRAQ7 to distinguish dead cells.  All DRAQ7(-) live cells were collected by using FACS (BD Biosciences, FACS Aria III). The cDNA library for NGS was conducted with Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (Single Index) or Next GEM single cell 3' Library & Gel beads kit ver 3.1 (Single Index) according to the manufacturer's instructions. scRNA-seq