GSM4253841: 049_DIOLs.S_d14_rep1; Mus musculus; RNA-Seq
For direct induction of DIOs, 5x10^4 ESCs were transferred into a low-cell-binding U-bottom 96-well plate (NUNC) in S10 medium (StemPro-34 SFM (Life Technologies) supplemented with 10% FCS, 150 μM ascorbic acid, 1× Glutamax, 1× penicillin/streptomycin and 55 μM 2-mercaptoethanol) with 150 ng/ml SCF, 0.5 µM of Shield1 (Clontech), and 10 nM Y-27632 (Wako). For the extended culture, we transferred the aggregates onto Transwell-COL membranes using a mouth pipet on day 2, and cultured them under normoxic conditions (20% O2 and 5% CO2 at 37 °C) for 2-4 weeks. For induction of fgDIOs and MII DIOs, 5x10^4 ESCs were aggregated with 30,000-75,000 E12.5 female gonadal somatic cells in S10 medium with 0.5 µM of Shield1 and cultured for 2 days, then transferred onto Transwell-COL membranes (Corning) soaked in S10 medium with 0.5 µM of Shield1 and cultured for 28 days. At 21-28 days of culture, individual follicles were manually isolated using sharpened tungsten needles. The isolated follicles were further differentiated by IVG for 7-11 days and IVM culture and fertilized by IVF as previously described  (Hikabe et al., 2016, Nature, Hayashi et al., 2017., Nat. Protcol) 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
GSM4253841