Extended Data Fig. 6: Generation of DNMT3A and 3B knockout mouse EpiSCs.
From: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers
a, Sequencing data showing the Dnmt3a and Dnmt3b mutations introduced into mouse DKO EpiSCs. Two different sgRNAs were used for each gene. Green box = PAM sequence, grey box = sgRNA sequence. Codons are colored black or blue, grey text denotes non-coding DNA. The respective single letter amino acid (aa) and protein sequence number is displayed above the DNA. b, Western blots showing protein levels for DNMT1, DNMT3A and DNMT3B in WT and DKO EpiSCs. PCNA is used as a loading control. Full blots in Source Data Fig. 2. Western blots were performed twice with consistent results. c, Violin plots display methylation levels for cDKO-DMRs and EpiSC DKO-DMRs in WT and DKO cells for each species. The UCSC LiftOver tool was used to map DKO-DMRs between species. Violin plots extend from the data minima to the maxima with the white dot indicating median, thick bar showing the interquartile range and thin bar showing 1.5x interquartile range. d, Schematic depicting how orthologous genes shared between human and mouse were assigned to the nearest DKO-DMR (left). Each gene was given a number (in kb) denoting the distance to the nearest DKO-DMR either up- or down-stream. Box plots for genes that were binned based in their distance to the nearest DKO-DMR in EpiSCs (shown in legend key), displaying the distance to the nearest DKO-DMR in human ESCs (right). A matched trend between distances in mouse and human cells in evident. For the box plots: the median is shown in bold, the box displays interquartile range and whiskers extend to 1.5 times the interquartile range. e, Venn diagram showing the overlap of orthologous genes that have a DKO-DMR within the gene body (0 kb away, as shown in panel d) in mouse EpiSCs and human ESCs. This overlap was highly significant (hypergeometric test).
