Amicon centrifugal filter device

A non-denaturing bisulfite treatment was carried out by incubating the IP-eluted chromatins with 460 μl of a freshly prepared solution of 5 M sodium bisulfite and 20 mM hydroquinone at 37°C overnight for 18 h (19 (link)). The following morning, the bisulfite-treated chromatin was washed 4 times with water using Amicon Ultra 10 K Centrifugal Filter Device (Millipore) followed by desulphonation in a 0.3 M NaOH solution for 20 min at room temperature. After removal of NaOH with an Amicon Centrifugal Filter Device by washing 4 times with TE buffer, the DNA was subjected a buffer exchange to 10 mM Tris pH 8.0, using Bio-spin P6 column (Bio-Rad, CA).

Non-denaturing bisulfite treatment was carried out by incubation of ssDNA (1 μg) or purified gDNA (5 μg) with a freshly prepared solution of 5 M sodium bisulfite and 20 mM hydroquinone at 37 °C. The presence of hydroquinone helps to protect DNA from degradation during bisulfite treatment (22 (link)). Following incubation for 18 h, bisulfite-treated DNAs were washed three times with water using Amicon Ultra 10 K Centrifugal Filter Device (Millipore) and desulphonated in 0.3 M NaOH solution for 20 min at room temperature. After removal of NaOH by the Amicon Centrifugal Filter Device, the DNA solution was buffer-exchanged to 10 mM Tris pH 8.0 using Bio-spin P6 column (Bio-Rad, CA, USA).