Supplementary Figure 3: Generation of Smchd1^GFP knock-in allele and its use in ChIP–seq.

a. acGFP-loxP-neomycin resistance-loxP was knocked in immediately before the stop codon of Smchd1 in 129T2/SvEms strain mESC, such that Smchd1 would be produced as a Smchd1-GFP fusion protein. Correctly targeted mESC clones were used to generate a line of mice, and the neomycin resistance cassette removed by Cre-mediated recombination. b. The Smchd1-GFP fusion protein was detected on the inactive X chromosome, as marked by H2aK119ub. GFP and H2AK119ub immunofluorescence performed in female Smchd1^GFP/GFP mouse embryonic fibroblasts. c. Optimisation of the ChIP-seq sonication conditions to release the Xi from the insoluble fraction. Sonication was performed in X₁₂₉^Xist∆AX_Castaneus NSCs. Screenshot from genome browser showing the X chromosome. Positions of genes are shown as black boxes along the top. Tracks displayed are showing normalised counts for reads that mapped to the inactive X chromosome (castaneus) minus the reads that mapped to the active X chromosome (domesticus). Height and colour of bar represents relative coverage. Sonication time is labelled on the left. As sonication time increases, reads that map to the Xi increase. Note that even after 30 minutes sonication, coverage from the Xi is still not as great as that from the Xa (relative coverage across the X chromosome is negative). Scale bar on right is read count. Data from n=1. d. Enrichment of GFP over WCE on Chromosome 2 and Chromosome 15. Height and colour of peaks indicates strength of read counts normalised to WCE; red is most enriched, blue is most depleted. HoxD locus on Chromosome 2 and HoxC locus on Chromosome 15 are marked with an asterisk. Position of MACS2-like peaks called using Seqmonk are indicated in blue.