Bisulfite conversion kit

DNA was extracted from 10⁶ cells (Qiagen DNeasy kit) according to the manufacturer’s protocol and quantified using the Picogreen reagent (Life Technologies). Bisulfite conversion was performed using the Bisulfite Conversion Kit from Zymo Research. Bisulfite converted DNA was labeled and hybridized to Infinium HumanMethylation450 BeadChips (Illumina, Inc.), scanned with an Illumina iScan BeadArray Scanner and quality controlled in GenomeStudio (Illumina, Inc.). Pathway analysis was performed using GREAT [17 (link)], and visualized using REVIGO [18 (link)].

The Illumina Infinium Methylation Assay (Illumina Inc., San Diego, CA, USA) was performed as per the manufacturer’s instructions. This assay covers 99% of RefSeq genes and 95% of Cytosine-guanine (CpG) islands in the human genome. The entire genome was interrogated and examined, but results with respect to UGT genes were the focus of this study. All CpG interrogation sites associated with all UGT genes were analyzed, but only significant results are presented. The DNA samples (1 μg) were first treated with sodium bisulfite using a Zymo Research Bisulfite conversion kit (Zymo Irvine, CA, USA) to deaminate un-methylated cytosine to produce uracil. Extraction, analysis, and visualization of the methylation data were performed using the Illumina Genome Studio software (version 2011.1) with methylation and genome viewer plug-ins (version 1.9.0, Illumina, San Diego, CA, USA). Differential methylation analysis was performed for grouped data utilizing the Illumina custom error model, resulting in a differential methylation score (diffscore) that provides directionality to the p-value. The diffscore was converted to an adjusted p-value with the formula: p-value = 1/((10^|DiffScore|)/10).