Fig. 3

Sonication of DNA generates a 5mC-dependent methylcytosine peak. a A methylcytosine peak, denoted mC*, was detected in the DNAs of eukaryotes which have high levels of 5mC (vertebrates and mammals) but undetectable in other organisms. Each bar represents the mean +/− standard error of the mean for 2–13 independent samples. b Representative UHPLC-ms/ms chromatograms displays the acquisition time of 5mC before and after sonication of human lymphoblastoid cell line (hLCL) genomic DNA. c mC* is detected at a later acquisition time than 4mC. Left panel depicts a zoomed in examination of hLCL genomic DNA with or without sonication from b) demonstrates a peak that appears at a later acquisition time than 4mC standard (lower panel). The inset displays boxplots of the distribution of retention times for 4mC standards (n = 15), mC* from DNA sonication of gDNA from several independent eukaryotic species (n = 6) and mC* detected in un-sonicated 5mC-containing DNAs from the same samples (n = 6). d Sonication of human DNA from a lymphoblastoid cell line (LCL), but not DNA from SF9 insect cells or C. elegans results in the generation of mC*. %mC* is shown in red and %5mC is shown in black. This graph represents the mean +/− standard error of the mean for 2 independent experiments. e Methylation of C. elegans or SF9 cell genomic DNA with the CpG C5-methyltransferase m.SssI followed by sonication is sufficient to generate mC*. %mC* is shown in red and %5mC is shown in black. This graph represents the mean +/− standard error of the mean for 2 independent experiments. *: p = 0.0136 by unpaired t test