Additional file 1 of KDM6A facilitates Xist upregulation at the onset of X inactivation

Additional file 1: Supplemental figure S1. CRISPR-Cas9 strategy and characteristics of Kdm6a KO cells. (A) Top: Schematic shows the location of the exonic deletion (Kdm6aΔE) that removed exons 2–4 of Kdm6a in female Tsix-stop ES cells and the promoter targeted deletion (Kdm6aΔP) made in female E8 ES cells. Exons are shown as vertical bars. The location of the PCR and RT-PCR primers (color-coded arrows) used to confirm each deletion is indicated. Below left: Images of gels after electrophoresis of PCR products using different sets of primers to confirm Kdm6a homozygous and heterozygous KO (- no deletion; + deletion positive). Actinβ was run as a control. Below right: Partial Kdm6a sequence obtained by Sanger sequencing as verification of deletions in Tsix-stop and E8 homozygous Kdm6a KO clones. Arrows point to the location of non-homologous end-joining and colored arrows correspond to those on the schematic. (B) Heat maps of gene expression differences between Tsix-stop wt (2 replicates, wt a and wt b) and KO clones (Tsix-Kdm6aΔEΔE17 and Tsix-Kdm6aΔEΔE21) at D0 and D15. Consistent with PCA clustering, more DEGs were identified at D15 than D0. Heat maps were generated using iDEP.95. (C) Scatter plots of expression of genes involved in germ layer differentiation obtained by RNA-seq. Tsix-stop wt and KO cells after differentiation (D15) show higher expression of germ layer-associated genes in wt versus KO cells (Tsix-Kdm6aΔEΔE17 and Tsix-Kdm6aΔEΔE21). Log2 average TPM values are from two wt replicates and two KO clones. E8-Kdm6a KO cells show no trend of significant difference in expressed germ layer genes at D2 or D4 of differentiation. P-values are calculated using 1-way ANOVA test.