Bisulfite Sequencing of DNA Methylation

Using the same DNA samples used in the BeadChip array, bisulfite reactions were performed using an EpiTect Bisulfite kit (Qiagen) with the following conditions: 95 °C for 5 min, 65 °C for 85 min, 95 °C for 5 min, and 65 °C for 175 min as previously reported [14 (link), 39 (link), 40 (link)]. The bisulfite converted DNA was amplified by PCR using the primer pairs shown in Additional file 3: Table S3 under the thermocycling conditions (95 °C for 10 min, and 40 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min followed by 10 min of final extension at 72 °C). The resulting products were cloned into pGEM-T easy vector (Promega, Tokyo, Japan). After sequencing reaction using a BigDye Terminator V3.1 kit (Applied Biosystems, Foster City, CA, USA), sequencing was performed with a 3130xl Genetic Analyzer (Applied Biosystems) as previously reported [24 (link), 41 (link)]. QUMA (http://quma.cdb.riken.jp/) was used to analyze the bisulfite sequencing data [42 (link)]. The bisulfite PCR primers are shown in Additional file 3: Table S3.