Supplementary Figure 7: Smchd1 deletion does not alter the compaction accessibility of the inactive X chromosome but does result in enriched H3K27me3 on the Xi.

a. Normalised Hi-C interaction frequencies at 100 kb resolution, generated from 200 million reads randomly subsampled from libraries at each condition, displayed as a heat map for the X chromosome in Smchd1^fl/fl (a) and Smchd1^del/del (b) NSCs. The colour of the contact map, from blue to red, indicates the log₂(contact frequency). c. ATAC-seq was performed in Smchd1^fl/fl and Smchd1^del/del NSCs (n=3 female, n=2 male), one week post Cre mediated deletion of Smchd1. Position of female specific X-linked differentially accessible peaks (DAP) from the ATAC-seq analysis called using and edgeR analysis on MACS2 peaks in red. Positions of genes are indicated in black along the top. d. Sex-specific differential analysis of the MACS2 peaks identified in all samples, found 540 female-specific DAPS genome-wide. These were regions that became more accessible in the absence of Smchd1 in females, but not in males, as shown by box plots showing the coverage at 36 differential X-linked ATAC-seq peaks in log (RPM) between female Smchd1^fl/fl (F) and Smchd1^del/del (D) NSCs (left, n=3), and males (right, n=2). The female specific DAPs were not enriched on the X chromosome relative to autosomes, X-linked DAPs comprising 6% of the total female specific DAP (36/540). p values shown were determined by Student’s two-tailed t test. e. Confocal imaging of Smchd1^fl/fl (top) and Smchd1^del/del (bottom) NSC nuclei following DNA FISH with X chromosome paint, n=23 observations per genotype, n=2 cell lines. DAPI stained DNA is shown (blue) and X chromosome paint (magenta), shown with 3D rendering performed in Imaris to measure volume. f. Widefield images of H3K27me3 immunofluorescence performed in female Smchd1^fl/fl (top) and Smchd1^del/del (bottom) NSCs (green). g. Confocal images with Airyscan processing of H3K27me3 immunofluorescence (red) in female Smchd1^+/+ (top) and Smchd1^{MommeD1/MommeD1} NSCs (bottom). Dot plots of volume and mean intensity of focal H3K27me3 enrichment in 3D using Imaris. Statistical comparisons were made using a two-tailed student’s t-test, line indicates mean, error bars are SEM. Measurements from 17 observations in n=2 cell lines, p <0.01. h. Genome browser tracks for the H3K27me3 ChIP-seq from ChrX:63,350,830-67,476,550 on the Castaneus-derived Xi chromosome in Smchd1^del/fl and Smchd1^del/del NSCs (n=2). Genes are shown in black, MACS2-called peaks in Smchd1^del/fl NSCs are shown in blue, and MACS2-called peaks in Smchd1^del/del NSCs are shown in red. Scale is normalised ChIP-seq read counts, normalised for read depth using the total autosomal read count.