GSM4253826: 034_d13_KO_Kdm1b_rep2; Mus musculus; RNA-Seq
IVD culture was performed as described previously (Hikabe et al., 2016, Nature). rOvaries (5,000 PGCLCs and 75,000 gonadal somatic cells) were placed on Transwell-COL membranes soaked in αMEM-based IVDi medium: αMEM supplemented with 2% FCS, 150 μM ascorbic acid (Sigma), 1× Glutamax, 1× penicillin/streptomycin and 55 μM 2-mercaptoethanol (Life Technologies). At 4 days of culture, the culture medium was changed to StemPro-34-based IVDi medium: StemPro-34 SFM (Life Technologies) supplemented with 10% FCS, 150 μM ascorbic acid, 1× Glutamax, 1× penicillin/streptomycin, and 55 μM 2-mercaptoethanol. From 7 days to 10 days of culture, 500 nM ICI182780 was added to the StemPro-34-based IVDi medium.Ovaries (in vivo) and rOvaries (in vitro) at each developmental stage were treated with CTK solution (0.1 mg/ml collagenase IV, 0.25% trypsin, 20% KSR and 1 mM CaCl2 in PBS) for 30 minutes at 37°C, then transferred into Accutase (Nacalai Tesque) and incubated at 37°C for 15 minutes. The IVD day11 or day13 SC+oocytes were isolated by FACS-sorting. For FACS preparation, the cells were dissociated by gentle pipetting and filtered by using a 70 μm-pored nylon mesh to remove cell clumps. Ovaries (in vivo) and rOvaries (in vitro) at each developmental stage were treated with CTK solution (0.1 mg/ml collagenase IV, 0.25% trypsin, 20% KSR and 1 mM CaCl2 in PBS) for 30 minutes at 37°C, then transferred into Accutase (Nacalai) and incubated at 37°C for 15 minutes. The IVD day11 or day13 SC+oocytes were isolated by FACS-sorting. For FACS preparation, the cells were dissociated by gentle pipetting and filtered by using a 70 μm-pored nylon mesh to remove cell clumps. Collected samples were transferred into a 1.5 ml DNA LoBind tube (Eppendorf). Directional RNA-seq libraries were prepared as described previously (Hamazaki et al., 2015). Briefly, polyA-tailed RNA were extracted by Dynabeads mRNA Purification Kit. Extracted polyA+RNA were converted to cDNA libraries by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina.
GEO Accession
GSM4253826