title: GSM5964685: RNA-seq morula Eed/Xist matDKO rep1; Mus musculus; RNA-Seq
library_construction_protocol: To generate F1 hybrid embryos, 8-12-week-old females of Eed fl/fl, Zp3-Cre/Eed fl/fl, Eed/Xist fl/fl, Zp3-Cre/Eed/Xist fl/fl were injected with 0.2 ml of CARD HyperOva followed by 7.5 I.U. of hCG at a 48 h interval. Cumulus–oocyte complexes (COCs) were harvested 15-17 hrs after hCG injection and inseminated with activated spermatozoa in HTF medium supplemented with 10 mg/ml bovine serum albumin (BSA, Merck Millipore 12657). Sperms were obtained from the caudal epididymis of adult PWK/PhJ mice [RBRC00213 (RIKEN Bioresource Research Center) originated from 003715 (the Jackson Laboratory)]. Spermatozoa were capacitated by 1 h incubation in CARD FERTIUP (Kyudo Co., Ltd., Japan). At 6 hrs post-insemination (hpi), zygotes with two pronuclei were transferred to KSOM and cultured in a humidified atmosphere with 5% CO2/95% air at 37.8°C. At 79 hpi, morula embryos were treated with Acidic Tyrode's solution to remove zona pellucida, washed in M2 media (Merck Millipore, MR-015-D), and then washed in 0.2% BSA/PBS. Five embryos per sample were pooled in Eed matKO and WT embryos, and 13 embryos per sample were pooled in Eed/Xist matDKO and WT embryos. RNA-seq libraries were prepared and sequenced as described previously (Mei et al. 2021). Briefly, cDNA was prepared by SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (TAKARA, 634890) according to the manufacturer's instructions. Two hundred picograms of cDNA were used for the library construction by using Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024) according to the manufacturer's instructions.