ChrRNA-seq of SPEN-SPOC and Polycomb mutant lines

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 the iXist-ChrX-Dom model cell line in which Xist on the M.m.Domesticus allele of female (XX) mESCs is under doxycyline-inducible control. Here we compare cell lines with engineered mutations to the SPEN SPOC domain or to components of the Polycomb system recruited by Xist (PCGF3/5 degron or deletion of Xist B/C-repeat), both individually and in combination. 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.