Following isolation of the DNA, bisulfite modification was performed for a 500-ng DNA sample in each case. The bisulfite conversions of DNA samples were conducted using the EZ DNA Methylation Kit (cat. no. #D5001; Zymo Research Corp.).
The genome-level methylation profiles of the modified DNA samples were investigated using the Infinium MethylationEPIC BeadChip Array on an iScan device (Illumina, Inc). The Infinium MethylationEPIC Array is a genome-wide DNA methylation analysis system based on bisulfite conversion and Infinium HD sequencing technology that queries differentiated loci using region-specific probes designed for methylated and non-methylated regions. The total methylation level for a queried locus is determined by calculating the ratio of fluorescent signals from the methylated and unmethylated regions (21 (link)). Data analyses of the experimental results were conducted using the Lumi libraries within the Illumina GenomeStudio v2011.1 Methylation Module v1.9.0 (https://www.illumina.com/techniques/microarrays/array-data-analysis-experimental-design/genomestudio.html) and R 3.0.2 (http://www.r-project.org). The differences in methylation for >850,000 regions on a point basis were investigated using this chip system. All six cases in the study group were evaluated at >850,000 different CpG points.