GSM7751806: WGBS W-L3 rep2; Bursaphelenchus xylophilus; Bisulfite-Seq
Protease K/phenol extraction for DNA extraction. 1 ug of total DNA was used for the construction of sequencing libraries. Prior to library construction, 1µg sample genome DNA mixed with unmethylated lambda DNA was fragmented using a Bioruptor system to a mean size of approximately 250bp. After fragmentation, the purified randomly fragmented DNA was subsequently treated with a mix of T4 DNA polymerase, Klenow Fragment and T4 polynucleotide kinase to repair, blunt and phosphorylate the ends. The bluntDNA fragments were subsequently 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 QIAquick PCR purificationkit (Qiagen). After that, 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, 10cycles 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. The PCR products were purified using QIAquick PCR purification kit (Qiagen). Before analysis with Illumina sequencing platform, the purified library was analyzed by the Bioanalyzer analysis system (Agilent, Santa Clara, USA) and quantified by the real time PCR.