GSM1463094: Normoxia (TET1-si) hMeDIP-seq; Homo sapiens; OTHER
Extraction was performed using TriZol and routine protocol. Genomic DNA was prepared using a genomic DNA extraction kit (QIAGEN, Germany) and sonicated with Bioruptor (Diagenode Inc. USA) to produce random fragments ranging in mean size from 300 to 1,000 bp. 2 µg of fragmented DNA was denatured for 10 min at 95°C and immunoprecipitated for 16 hr at 4°C with 5 µl of 5-methylcytidine antibody (Eurogentec, Germany) in a final volume of 250 µl IP buffer (150 mM NaCl, 1mM EDTA, 1mM EGTA, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 0.05% Triton X-100). The mixture was incubated with 50 µl protein A beads for another 4 hr at 4°C and washed three times with 1 ml of IP buffer. Beads were resuspended with 500 µl digestion buffer (50 mM Tris [pH 8.0], 10 mM EDTA, 1% SDS) containing 10µl proteinase K (20 mg/ml stock) overnight at 65°C. D