Sodium bisulfite treatment

The Bisulfite conversion was carried out according to the protocol of Clark et al. (1994), with minor modifications. Briefly, 1-2 μg DNA in a volume of 50 μL was denatured by adding 3 μL of 5 M NaOH and incubating for 15 minutes at 37°C. After denaturation, 420 μL of 3.9 M (saturated) sodium bisulfite (Sigma, final concentration 3.4 M; pH 5.0) and 33 μL of 20 mM hydroquinone (Sigma, final concentration 0.58 mM) were added to the denatured DNA and incubated at 50°C for 12-14 h. Treated DNA was desalted and purified using the Wizard DNA Clean-Up system (Promega, USA), desulfonated by adding 3 μL of 5 M freshly prepared NaOH, followed by incubation for 15 minutes at 37°C and finally precipitated with 1 μL of glycogen (Fermentas, final concentration 150 μg/mL, 25 μL of 10 M ammonium acetate (pH 7.0) and 150 μL of chilled absolute ethanol. The precipitated DNA was pelleted and resuspended in 40 μL of sterile water and stored at -20 °C until used.