GSM5702680: MCF7_Input_E2_10d; Homo sapiens; OTHER
gDNA was sonicated to 200–500 bp fragments, denatured in boiling water and immediately cooled on ice. Fragmented DNA was added to IP buffer (10 mM Na-Phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100). 5hmC/ 5mC antibody (5hmC antibody, Active Motif #39769; 5mC antibody, Active Motif #39649) was added to the sample and incubated at 4°C for 4 h. Protein A/G beads were added to each reaction and incubated at 4°C for 2 h. The beads were washed using IP buffer for three times. The sample was resuspended in Proteinase K digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS, included 2 μl protease K) and incubated at 50°C for 2 h. The final sample was extracted using QIAquick PCR Purification Kit.  hMeDIP libraries were prepared for sequencing using standard Illumina protocols.