Extended Data Fig. 1: Validation and characterization of PKO mESC.

a, Schematic of Tet1/2/3 and Dnmt3a/3b knockout strategies. b, Western blot of TET1/2 and DNMT3A/3B proteins (left) and dot blot results of 5mC and 5hmC (right) in WT, TKO and PKO mESCs. TET3 is not expressed in mESCs. Western blot images were cropped (see Extended Data Fig. 10). c, The Sanger sequencing results for Tet mutations. d, Bar charts showing numbers of TKO-WT, PKO-TKO and PKO-WT DMRs. e, Bar charts showing the enriched gene ontology (GO) terms for genes associated with either promoter or distal TKO-WT DMRs (left). Similar analysis results are shown for PKO-TKO DMRs (right). P-values (Fisher’s exact test, two-sided, not adjusted by multiple comparisons) are transformed by log10. f, Boxplots showing the gene expression changes (log2 ratio of RPKM) for those associated with TKO-WT or PKO-WT DMRs (n = 2 biologically independent experiments). The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times of the IQR, respectively. g, Bar charts showing cell numbers of WT, TKO and PKO mESCs upon cell propagation (n = 3 biologically independent experiments. Data are presented as mean values + /- SD). h, Bar charts showing the pluripotency gene expression in WT, TKO and PKO mESCs.