Loss of chromatin accessibility at some CTCF binding sites correlates with reduced CTCF binding upon ESC differentiation. (A) Differences in RE accessibility at specific DHSs were confirmed by qPCR across an RS of interest at each locus. Remaining uncut DNA after RE digestion of each indicated KD is shown for several DHSs that exhibited accessibility differences by RED-seq. Data are normalized to uncut genomic DNA. (B) Confirmation of restriction enzyme accessibility surrounding CTCF binding sites, as in (A). (C) CTCF ChIP-qPCR data are shown for the indicated KDs at several CTCF binding sites. Controls are CTCF binding sites in which accessibility did not change upon Oct4 KD. Data are presented as a percentage of input DNA. Shown are the mean ± SD of three technical replicates from one representative experiment of two biological replicates performed. (D-E) RED-seq data (D) and MNase-seq data (E) over Klf4 binding sites, plotted as in Figure 3. (F) Average accessibilities over DHSs, CTCF binding sites, and Klf4 binding sites were quantified for biological replicate KD experiments from –200 to +200 bp with respect to the indicated feature. P-values indicating statistical significance of accessibility between EGFP KD and Oct4 KD are indicated.