Genomic DNA was extracted by lysing the cells (20 mM Tris-HCl pH 8.0, 4mM EDTA, 20mM NaCl, 1% SDS), purification of nucleic acids by phenol-chloroform extraction followed by precipitation with Na Acetate, resuspension in TrisEDTA and RNase A treatment. DNA was sonicated with Covaris (Peak Incident power 140, Duty factor 10%, cycles per burst 200, treatment time 100s) to obtain 200-500bp fragments. Sonicated DNA was then subjected to end repairing, A-tailing and adapter ligation, denatured and immunoprecipitated with antibodies against 5hmC (Active Motif, #39769) overnight at 4°C in IP buffer (100nM Na-Phosphate buffer pH 7.0, 1.4M NaCl, 0.5 % Triton X-100). DNA was purified for library size-selection and amplification. Single-end libraries were prepared and sequenced in 100bp reads runs according to Illumina instructions. The steps of end repairing, A-tailing and adapter ligation were performed before denaturation and immunoprecipitation while size selection and library amplification were performed after
GEO Accession
GSM2522709