GSM8459875: Ctrl3 [RRBS]; Bos taurus; Bisulfite-Seq
DNA was extracted from blood samples of pig using a DNA extraction Kit(Beijing, China, Tiangen Biochemical Technology Co., Ltd., DP304-03) About 1µg 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 50µl consisting of 20µl adapter ligated DNA, 4µl 2.5mM dNTP, 5µl 10×buffer, 0.5µl JumpStart™ Taq DNA Polymerase, 2µl PCR primers and 18.5µl water and the following thermal cycling program was 94°C 1 min, 12cycles of 94 °C 10s, 62°C 30s, 72°C 30 then prolong with 5min at 72°C and products could be hold at 12°C. 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.