Methylation Analysis by Pyrosequencing

Cells were seeded in T75 culture flasks and treated as described above with 10 μM 5AZA. DNA was extracted and bisulfite converted, as described above. Pyrosequencing assays were designed using the PyroQ assay design software. A common tag was placed on either the forward or reverse primer (depending on the strand to be sequenced) and a common universal biotinylated primer was used for all reactions as previously described [27 (link)]. PCR was performed using a nested PCR for specific amplification and cycling conditions included denaturation at 95°C for 4 min, followed by 10 cycles of 94°C for 15 s, touchdown from 60°C to 50°C (−1°/cycle) for 15 s and 72°C for 20 s, followed by a further 30 cycles at 50°C annealing temperature. The second PCR used 2 μl of a 1:10 dilution of the first PCR as template and the same cycling conditions. All products were confirmed to be single bands by agarose gel electrophoresis. Methylation values were calculated as an average of all 10 CpG sites within each assay as determined by the Pyro Q-CpG Software (Biotage). Primers detailed in Supplementary Table S2.