title: WTWT002
design: About 1g of genomic DNA mixed with unmethylated lambda DNA was digested by MspI enzyme for 16 hours at 37. After digestion, the libraries were constructed as the Illumina Pair-End protocol with some modifications. Briefly, purified digested DNA was subsequently treated with a mix of T4 DNA polymerase, Klenow Fragment and T4 polynucleotide kinase to repair, blunt and phosphorylate ends. After that, the blunt DNA were 3 adenylated using Klenow Fragment (3-5 exo-) and following with ligation to adaptors synthesized with 5-methylcytosine instead of cytosine using T4 DNA Ligase. After each step, the DNA was purified using MinElute PCR Purification Kit (Qiagen). ZYMO EZ DNA Methylation-Gold Kit was employed to convert unmethylated cytosine into uracil according to the instructions. Finally, PCR was carried out in a final reaction volume of 50l consisting of 20l adapter ligated DNA, 4l 2.5mM dNTP, 5l 10buffer, 0.5l JumpStart Taq DNA Polymerase, 2l PCR primers and 18.5l water and the following thermal cycling program was 94C 1 min, 12cycles of 94 C 10s, 62C 30s, 72C 30 then prolong with 5min at 72C and products could be hold at 12C. Before analysis with Illumina sequencing platform, the size selected library was analyzed by the Bioanalyzer analysis system (Agilent, Santa Clara, USA) and quantified by the real time PCR.