DNA methylation was analyzed on bisulfite-converted genomic DNA (EZ DNA Methylation Kit; Zymo Research) using the MassARRAY system (Sequenom) as reported24 (link). Quantification of DNA methylation levels were calculated by the Epityper software v.1.2 (Sequenom). Primers are listed (Supplementary Table 2). For 5-hydroxymethylcytosine measurements, the assay was performed as reported9 (link),25 (link) with minor modifications. In brief, 250 ng genomic DNA was glycosylated with 4 U T4 phage beta-glucosyltransferase (T4-BGT) in 50 μl glycosylation buffer (50 mM potassium acetate, 20 mM Tris-acetate [pH 7.9], 10 mM magnesium acetate, 1 mM DTT, 100 mM uridine diphosphoglucose (NEB) or left untreated. After incubation for 12 h at 37°C, T4-BGT was inactivated by incubation for 10 min at 75°C. DNA was digested with MspI (2 U, 6 h at 37°C), purified and analyzed by qPCR. To normalize for digestion efficiency, the amount of amplicon corresponding to the MspI site (CpG#7) at TCF21 promoter was normalized to the amplicon corresponding to the single MspI site on CpG island 2. Relative levels of 5-hydromethylcytosine were calculated as ratio of T4-BGT treated- versus non-treated samples.