Bisulfite conversion of DNA was performed
using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA),
and the resultant product was suspended in 40 μL of distilled
water. The methylation status of the PCR product was quantitatively
determined using pyrosequencing, as previously described.6 (link),9 (link) Briefly, the biotinylated PCR product was bound to Streptavidin
Sepharose High Performance beads (GE Healthcare Life Sciences, Little
Chalfont, UK), washed, and denatured using a 0.2 M NaOH solution.
After the addition of 300 nM sequencing primer to the purified single-stranded
PCR product, pyrosequencing was carried out on a PyroMark Q24 MD System
(Qiagen) with the Pyro Q-CpG software (Qiagen) according to the manufacturer’s
instructions. Primer sequences and conditions, as well as the number
of analyzed CpG sites, are shown inTable S2 . Methylation control samples (0 and 100% methylated)51 (link),52 (link) were used for confirmation in every assay.
using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA),
and the resultant product was suspended in 40 μL of distilled
water. The methylation status of the PCR product was quantitatively
determined using pyrosequencing, as previously described.6 (link),9 (link) Briefly, the biotinylated PCR product was bound to Streptavidin
Sepharose High Performance beads (GE Healthcare Life Sciences, Little
Chalfont, UK), washed, and denatured using a 0.2 M NaOH solution.
After the addition of 300 nM sequencing primer to the purified single-stranded
PCR product, pyrosequencing was carried out on a PyroMark Q24 MD System
(Qiagen) with the Pyro Q-CpG software (Qiagen) according to the manufacturer’s
instructions. Primer sequences and conditions, as well as the number
of analyzed CpG sites, are shown in
