RNA-seq in differentiating female FVBxCAST F1 ES cells with Smarca4 gene knockdown [Female Smarca4 RNA-seq]

Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation. This experiment is designed to test if X chromosome inactivation is altered upon Smarca4 gene knockdown. Overall design: n=1 Smarca4 knockdown for two different hairpins, n=1 for matched nonsilencing controls. Samples taken at day 0 and 6 of FVBxCAST F1 ES cell differentation