Hydroxymethylation profiling by hMeDIP-seq

DNA was sonicated with Covaris to produce fragments in size range of 300–500 bp. Prior to hMeDIP procedure, Illumina adaptors were ligated to (5 × 1 μg) of fragmented DNA as described in the TruSeq LT DNA Sample Preparation kit, Illumina. The hMeDIP assay was performed according to the manufacturer’s instructions (Active Motif, hMeDIP, Cat No 55010). Briefly, 3 × 1 μg of fragmented adapter-ligated DNA was spiked with 50 ng of either unmethylated, 5mC methylated or 5hmC hydroxymethylated 338-bp PCR product of APC genomic locus. The DNA was denatured for 10 min at 95 °C and immunoprecipitated overnight at 4 °C with 4 μl of 5hmC polyclonal antibody (Active Motif Cat No 55010). To allow selective enrichment of immune-captured DNA fragments, the mixture was incubated with 25 μl of Protein G magnetic beads for 2 h at 4 °C prior to washing all unbound DNA fragments. The bound hydroxymethylated DNA was eluted, treated with proteinase K and purified by Phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. The specificity of the hMeDIP assay was validated by qPCR of the unmethylated, methylated and hydroxymethylated spike-in APC controls and dot blots.