SRP341230 PRJNA770992 GSE185843 ChrRNA-seq of mESC to NPC timecourses for WT and SmcHD1 KO clones 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 compare WT cell lines over timecourses of ES to NPC differentiation (under constant Xist induction), with SmcHD1 KO clones cultured in parallel. 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. In later timepoints, SmcHD1 KO clones fail to complete gene silencing for a subset of genes, demonstrating a role for SmcHD1 in the establishment phase of XCI. GSE185843 pubmed 35584662 parent_bioproject PRJNA771073