Hiseq benchtop sequencer

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)}

The DNA methylation level of the target CpG site is defined as the number of methylated reads at that site (i.e., the number of reads with base C detected) divided by the total number of reads at that site, and was obtained by MethylTarget sequencing (Genesky Biotechnologies Inc. Shanghai, China), a method based on next-generation sequencing-based multiple targeted CpG methylation analysis. Primer design and validation were performed using bisulfate-converted DNA samples on the Methylation Primer software. The primer sets were designed to flank each target CpG site by 100–300 nucleotides and are summarized in Supplementary Table S1. After PCR amplification (HotStarTaq polymerase kit, TAKARA, Tokyo, Japan) and library construction, paired-end sequencing was performed (Illumina Hiseq Benchtop Sequencer, CA, USA) in accordance with the manufacturer’s protocol.