Validating WGBS Data via DMR Sequencing

To validate the WGBS data, we randomly selected six DMRs for BSP [54 (link)]. Genomic DNA samples from all TC, TT, SC, and ST replicates were first bisulfite-converted using EZ DNA Methylation-Gold kits. The bisulfite-converted DNA samples were then PCR amplified as previously described using bisulfite-specific primers (Additional file 1: Table S4), which were designed using Methyl Primer Express v1.0 (Applied Biosystems, USA). The PCR products were separated using 1% agarose gel electrophoresis. Target DNA fragments were then extracted and gel-purified using QIAEX II Gel Extraction Kits (Qiagen, Germany). The purified DNA was ligated with the pGEM-T Easy vector (Promega, USA) and cloned in DH5α cells (Takara, Japan). For each sample, 10 positive clones were selected and sequenced individually using Sanger sequencing.