Additional file 1.

Figure S1. (a) Dot blot comparing the specificities and sensitivities of two commercially available 5hmC antibodies. Antibodies were tested over a dilution gradient of genomic liver DNA (200 ng/μl to 2.5 ng/μl) as well as for their relative affinities against PCR products synthesized in the presence of either dCTP, dmCTP or d5hmCTP [68]. Although both antibodies can distinguish 5hmC from 5mC and non-modified C (see controls), only the polyclonal (pAB) α-rabbit antibody (Active motif) can detect 5hmC in genomic mouse liver DNA. It must be noted that the relative levels of 5hmC present in the control PCRs are far greater than that in the genomic DNA. (b) The HmeDIP technique is highly specific for 5hmC-modified DNA relative to unmodified or methylated DNA. qPCR was run on immunoprecipitated material that had been spiked with a 5hmC-rich DNA sequence. Enrichment of the 5hmC rich control DNA was only observed when immunoprecipitated with the 5hmC antibody and not with an antibodies against 5mC, non-modified C or IgG.

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Thomson et al. Genome Biology 2012 13:R93   doi:10.1186/gb-2012-13-10-r93