Genome-wide DNA Methylation Analysis

Following bisulfite treatment of 1 μg genomic DNA using the EZ DNA Methylation kit (Zymo Research, Irvine, CA), the bisulfite-converted DNA was hybridized onto the Infinium Methylation EPIC BeadChip (Illumina, San Diego, CA), following the Illumina Infinium HD Methylation protocol in the Genomics Core Facility at UAMS. The Methylation EPIC BeadChip covers >850000 CpG sites, and has increased genome coverage of regulatory regions and higher reproducibility and reliability compared to previous versions^{15 (link)}. Whole genome amplification, hybridization, staining and scanning steps for all samples were performed, the Illumina iScan SQ scanner was used to create images of the single arrays, and the intensities of the images were extracted using the Methylation module (v.1.9.0) of the GenomeStudio (v.2011.1) software (Illumina). Raw intensity data as IDAT files were imported into the ChAMP R package^{11 (link)} for the processing and analysis of the methylation arrays. The BMIQ algorithm was used in the normalization of the data. Probes on a blacklist of probes that are known to be cross-reactive were removed.