GB2 hMeDIP
Experimental Design:  Cells were digested with proteinase K and RNase A, and genomic DNA was purified by phenol/chloroform extraction. Purified genomic DNA was sonicated to 200-500 bp with a Handy Sonic (TOMY). hMeDIP was performed as described previously (Weber et al., 2005) with minor modifications. Briefly, 4 μg of fragmented genomic DNA was immunoprecipitated with 4 μl of polyclonal antibody against 5hmC (Active Motif) at 4 ̊C overnight in a 500 μl of IP buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The mixture was incubated with 30 μl of Dynabeads Protein G at 4 ̊C for 2 h, and washed 3 times with 1 ml of IP buffer. The beads were suspended in 20 μg of proteinase K and incubated at 55 ̊C for at least 3 h. Immunoprecipitated DNA was purified by phenol/chloroform extraction followed by isopropanol precipitation.   Library Construction:  hMeDIP fractions were ligated to sequencing adapters and amplified according to the manufacturer’s instructions (BioLabs NEB Next ChIP-Seq Library Prep Reagent Set for Illumina).