Targeted CpG Methylation Analysis by MethylTarget Sequencing

DNA methylation at specific CpG sites was determined by MethylTarget sequencing (Genesky Biotechnologies Inc., Shanghai, China), using next-generation sequencing-based multiple targeted CpG methylation analysis.^{41 (link),42 (link)} Primer design and validation were performed using Methylation Primer software on bisulfate-converted DNA. Primer sets were designed flanking each targeted CpG site in a 100–300 nucleotide region and are shown in Supplementary Table 3. Genomic DNA was extracted from frozen samples using Genomic Tip-500 columns (Qiagen, Valencia, CA, USA) and from bisulfite-converted samples using an EZ DNA Methylation™-GOLD Kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. After PCR amplification (HotStarTaq polymerase kit, TAKARA, Tokyo, Japan) and library construction, samples were sequenced (Illumina HiSeq Benchtop Sequencer, CA, USA) by paired-end sequencing according to the manufacturer’s guidelines.^{43 (link)}