meQTL analysis

We used R software MatrixeQTL package32 (version 2.15.1; R Project for Statistical Computing, Vienna, Austria) for eQTL analysis. Each methylated CpG level was regarded as a continuous variable rather than a discrete variable. Correlations between the methylation level of each CpG site and each gene were evaluated using MatrixeQTL. As DNA methylation mostly influences the expression of genes via promoter regions, we distinguished cis- and trans-regulation for further analysis. The gene and methylation CpG sites were extracted from Illumina Human Infilium 450k BeadChip annotation files and University of California, Santa Cruz (UCSC) reference gene list locations. For reference genes, the gene location was counted from the transcription start site to terminal site. We set a P value threshold of 1 × 10⁻⁵ for cis-regulated and 1 × 10⁻⁶ for trans-regulated eQTLs. Cis-regulated meQTLs were defined as interacting pairs with tested DNA methylation sites and genes <1 megabase (MB), whereas trans-regulated meQTLs were defined as pairs with genes >1 MB or located on other chromosomes.