Fig. 5
Setdb1-mediated H3K9 methylation is required for silencing at hundreds of genes spread along the inactive X chromosome. a Schematic representation of these RNA-seq experiments. Female Xist ∆A/+ 129/CAST ES cells, where the Xist ∆A allele forces the CAST-derived X to be the obligate Xi, were differentiated by transitioning them from 100 % 2i plus LIF media into differentiation media in 4 increments over 4 days (see “Methods” section for full details). RNA was collected on day 0 and differentiation induced. Cells were transduced the following day with shSetdb1.6 or Nons control, puromycin selection for transduced cells was performed the following day, then RNA collected at day 3 and day 5 post-differentiation induction. In this system, XCI can be observed as a decrease in exonic reads mapping across SNPs to the CAST X compared to the 129 X; i.e. a decrease in the Xi/Xa ratio. b, c Box plots showing the Xi/Xa ratio over the differentiation time course for the Nons sample, suggesting that XCI is occurring as expected in both replicates 1 (b, n = 1221–1769 SNPs) and 2 (c, n = 874–1649 SNPs). d, e The Xi/Xa ratio is plotted for replicates 1 (d, n = 1033–1808 SNPs) and 2 (e, n = 874–1696 SNPs) at day 3 and day 5 of differentiation for cells transduced with shSetdb1.4, shSetdb1.6 or Nons. For b–e, p values are shown (Student’s two-tailed t test). f Venn diagram showing genes containing at least one reactivating SNP in replicates 1 and 2. Reactivating SNPs are defined as having a higher Xi/Xa ratio in both the shSetdb1.4 and shSetdb1.6 samples compared to the Nons. g Location on the Xi of genes carrying reactivating SNPs, derived from a Seqmonk browser track (www.bioinformatics.babraham.ac.uk/projects/seqmonk/). h–l RNA FISH stained for Xist (red) and DAPI (blue) in freshly derived day 5 differentiated female Bl/6 ES cells, following treatment with a negative control Nons (h), positive control shHnrnpu (i), shSetdb1.4 (j) or shSetdb1.6 (k). l Quantification of Xist clouds observed by FISH. See also Additional files 12: Figure S11, 14: Figure S12