DNA-seq in male ES cells cultured by either standard ES cell culture or our improved culture conditions [Male DNA-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 the effect of our improved ES culture conditions on the male ES cell karyotype. Overall design: n=2 for male cells cultured in standard culture conditions and n=2 for male ES cells cultured in our improved conditions. Samples taken at passages 0, 10 and 20 of ES cell maintenance in 2i media.