Fig. 1: Transcriptome-wide distribution of 5hmC in ESCs and EBs.

a Specificity of 5hmC antibody. Only the 5hmC-modified transcript shows enrichment after hMeRIP compared to the controls. Unmodified, 5mC-modified, and 5hmC-modified transcripts (IVT: in vitro transcribed) were used to spike total RNA prior to hMeRIP-qPCR. Data are means ± SEM (n = 2 independent experiments). b hMeRIP-Seq in WT ESCs reveals the presence of 5hmC within many transcripts (n = 3). Experiments were performed in biological triplicate and results were normalized as described in the “Methods” section. c Exemplative hMeRIP-Seq profiles of Cdh4 and Mdc1 in WT ESCs with their corresponding input control tracks (IGV tracks) (red frame shows peak location). d Bar chart showing the distribution of 5hmC peaks according to the type of structural element within transcripts, next to the expected distribution. e Top sequence motif identified in the centers of 5hmC peaks (E-value < 2.2e−117). f 5hmC is found in many key pluripotency-related mRNAs. Comparison of the above hMeRIP-Seq data sets with publicly available mouse ESC data sets^{10,11,12,13,14}, with representative examples of known transcripts encoding ESC core pluripotency regulators such as Eed, Jarid2, Smarcc1, Paf1, and Mbd3. g Scheme of the previously reported protocol³⁶ used for spontaneous differentiation of ESCs into EBs upon LIF removal, as described in the Methods section, with their relative Tet expression levels as measured by RT-qPCR. Data are means ± SEM (n = 3 independent experiments). h Decreased global 5hmC during spontaneous differentiation, as assessed by dot blotting. Data are means ± SEM (representative blot from three independent experiments, two-tailed Student’s t-test). i Many pluripotency-related transcripts show reduced 5hmC during differentiation. Left: Box plot for hMeRIP-Seq ESCs and EBs (n = 3 independent experiments, two-tailed Student’s t-test). In the box plot, the boxes represent the interquartile range of the records, and the lines across the boxes indicate the median value of the records. The whiskers indicate the highest and lowest values among the records that are no more than 1.5 times greater than the interquartile range. The range between notches represents the 95% confidence interval. Right: Pie chart highlighting the percentage of transcripts showing reduced 5hmC marking, 72 of which are identified as pluripotency-promoting mRNAs. j Exemplative hMeRIP-Seq profiles of Eed and Jarid2 in ESCs vs EBs (IGV tracks) (red frame shows peak location). Source data are provided as a Source Data File.