Fig. 2

UHPLC-ms/ms quantification of 6mA and 5mC DNA methylation in eukaryotes. UHPLC-ms/ms quantification of a 6mA and c 5mC in 16 eukaryotic species and 2 bacterial strains. Phylogram displayed below represents the evolutionary distance between species. Non-gnotobiotic mammalian and G. gallus samples were extracted from brains, R. temporaria samples were extracted from liver, D. rerio samples were extracted from the posterior end of adults, C. elegans were extracted from bleached embryos or young adults, the E. coli represent two different K12 strains; wild-type OP501 and dam⁻dcm⁻ (NEB C2925). Each bar represents the mean +/− standard error of the mean for 2–20 independent samples except for single samples for R. temporaria, G. gallus, O. aries, R. norvegicus, C. porcellus, B. Taurus, and M. mulatta. We note that in several UHPLC-ms/ms experiments 6mA was below our limit of detection (< 0.00005%) in metazoan DNAs (data not shown). b A heat map of the 6mA and 5mC quantifications and calculated values demonstrates 6mA is rare, if present at all, relative to 5mC in metazoan. d Chromatin immunoprecipitation of 293 T gDNA with a histone H3 antibody shows no significant depletion of 6mA levels as assessed by UHPLC-ms/ms. Each bar represents the mean +/− SEM of 3 independent experiments of 1–3 replicates. ns: not significant as assessed by Welch’s t test