We used bisulfite pyrosequencing for methylation analyses of target promoter regions. Each primer was designed using the PSQ assay design program (Qiagen) (sequences are listed in Supplementary Table 4 ). PCR reactions were performed in a volume of 20 μL with 20 ng or less of bisulfite-converted gDNA, 10 μL 2×Hot/Start PCR premix (Enzynomics, Daejeon, Korea), 1 μL forward primer (10 pmole/μL), and 1 μL biotinylated-reverse primer (10 pmole/μL). Amplifications were performed according to general pyrosequencing guidelines involving denaturing at 95°C for 10 minutes, followed by 50 cycles at 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and a final extension of 72°C for 10 minutes. The PCR reaction (2 μL) was confirmed by electrophoresis in a 2.5% agarose gel and visualized by ethidium bromide staining.
The ssDNA template was prepared from 16 to 18 μL of biotinylated PCR product using streptavidin Sepharose HP beads (Amersham Biosciences, Piscataway, NJ, USA), following the PSQ 96 sample preparation guide. Sequencing primers (15 pM) were then added for analyses. Sequencing was performed on a PyroMark ID system using the Pyro Gold reagent kit (Biotage, Uppsala, Sweden), per manufacturer’s instructions. The analyzed sequences are listed inSupplementary Table 4 .
The ssDNA template was prepared from 16 to 18 μL of biotinylated PCR product using streptavidin Sepharose HP beads (Amersham Biosciences, Piscataway, NJ, USA), following the PSQ 96 sample preparation guide. Sequencing primers (15 pM) were then added for analyses. Sequencing was performed on a PyroMark ID system using the Pyro Gold reagent kit (Biotage, Uppsala, Sweden), per manufacturer’s instructions. The analyzed sequences are listed in