Supplementary Figure 9: Transcriptional regulation of Rspo1 and Spry1 in PRDM15-binding-site-mutated clones.

(a) qPCR analysis of Rspo1 expression in WT versus mutant clone R15 (left) and in Prdm15^fl/fl versus Prdm15^Δ/Δ ESCs; expression was normalized to Ubiquitin and WT cells were set as the reference. The sequences of the PRDM15-binding site in WT and R15 clones are shown to the right. (b) Similar to a, Spry1 expression levels are analyzed by qPCR in three single clones targeted with CRISPR–CAS9 (using three different gRNAs). Genotypes and sequences of the CRISPR–CAS9-targeted region in WT clone and CRISPR clones S16, S19 and S24 are shown to the right; note the disruption within the consensus motif sequence in clones S19 and S24 and upstream of it in clone S16, which is used as the control for further experiments. In a and b, data are from four independent experiments; center values, mean; error bars, s.d. (c) ChIP–qPCR analysis of PRDM15 and RNAPII binding along with H3k27ac, H3k27me3 and total histone H3 enrichment on the promoter region of Spry1 in WT versus S16 clones. Data are from one representative experiment (n = 2) and are averaged across two technical replicates ± s.d. Student’s t test (two-sided) was used.