CpG methylation analysis by bisulfite sequencing

Genomic DNA was prepared using the NucleoSpin Tissue XS kit (Macherey-Nagel). After sodium bisulfite treatment (MethylEasy Xceed, Human Genetic Signatures), modified DNA was amplified by PCR and subcloned into PCR2.1-TOPO Vector (Invitrogen). PCR primers used were 5′-TTG​GGT​TAA​GTT​TGT​TGT​AGG​ATA​G-3′ and 5′-ATC​TAA​ACC​CTA​TTA​TCA​CAA​CCC​C-3′. The colonies (16–48 colonies/region) were directly amplified using the Illustra TempliPhi Amplification Kit (GE Healthcare) and sequenced.