Genome-Wide DNA Methylation Analysis

Genomic DNA was extracted as described above. Site-specific CpG methylation was analyzed by means of the Infinium^® HumanMethylation27 bead-array-based technique. This array was developed to assay 27,578 CpG sites selected from over 14,000 genes. The Zymo EZ DNA Methylation KitTM (Zymo Research, Orange, USA) was used to treat 1 μg genomic DNA with sodium bisulfite. This was done according to the manufacturer's procedure, with the alternative incubation conditions recommended when using the Illumina Infinium^® Methylation Assay. The assay was performed on 4 μL converted gDNA at 50 ng/μL concentration, according to the protocol described in the Infinium^® Methylation Assay Manual. Raw Infinium data were filtered by removing low quality data using a detection P-value threshold of 0.05. As the vast majority of Illumina HumanMethylation27 targets are covered by the HumanMethylation450 we used the extended annotation provided by Price et al. [78 (link)] to filter out the Probes containing SNPs (see [11 (link)] for a detailed description). Probes associated to X and Y chromosomes were removed from the analysis. β-values were computed using the formula β-value = M/[U + M] where M and U are the raw “methylated” and “unmethylated” signals, respectively.