ChrRNA-seq after 10 days of Xist induction in mESCs

X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. Making use of a SPEN separation-of-function mutation we show that SPEN and Polycomb pathways in fact function in parallel to establish gene silencing. Additionally, we find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Overall design: This series contains datasets of Chromatin RNA-seq experiments conducted in both iXist-ChrX-Dom and iXist-ChrX-Cast model cell lines, in which Xist is dox-inducible on the M.m.Domesticus or M.m.Castaneous allele of female (XX) mESCs. Here we investigate silencing in mESCs after continuous Dox-induced Xist expression for 10 days. We analyze XCI by isolation of chromatin-associated RNA (ChrRNA) for next-generation sequencing, followed by allelic assignment of reads to Xi or Xa chromosomes, and thus calculation of allelic ratio (Xi/(Xi+Xa)) as a quantitative measure of X-linked silencing on a gene-by-gene basis. Silencing does reach completion in mESCs, demonstrating that differentiation-dependent mechanisms are required to complete XCI.