iM-Seq Protocol for Methylated DNA Detection

Synthesized DNA oligoes corresponding to pH 5.5/7.0 biased peaks, synthesized DNA oligoes with methylated C at different positions, genomic DNA, zebularine- and M.Cvip I-treated genomic DNA were denatured and re-associated in iM reconstruction buffer (50 mM Tris–AcOH, pH 5.5 or 7.0) at 95°C for 8 min. Denatured and reassociated DNA was loaded on Amersham Hybond-N⁺-nylon membrane followed by pre-blocking in 5% milk for 45 min at RT. The pre-blocked membrane was incubated with the iMab antibody in the standard blotting or genomic IP buffer with pH 5.5 or 7.0 overnight at 4°C, then incubated with anti-IgG (HRP) antibody for an additional 1.5 h. The procedures for immune-signal development were the same as our previous procedures (71 (link)). Each blot was repeated at least two times for signal quantification.