GSM2344434: CON-37; Sus scrofa; Bisulfite-Seq
Piglets were euthanized by sodium pentobarbital (200 mg/kg, i.a.). The gastrointestinal tract was immediately removed. The small intestine was carefully emptied of its contents and weighed. From the middle of distal small intestine, small pieces were snap frozen in liquid nitrogen and kept at −80°C for later analysis of DNA methylome and inflammatory cytokines. Genomic DNA from the above 14 distal intestinal tissues was extracted using DNeasy Blood & Tissue Kit (Qiagen) and was subjected to RRBS library preparation. Briefly, 1.5 µg of genomic DNA was digested with MspI enzymes (NEB), followed by end-repairing, A-base tailing and 5-methylcytosine modified adapter ligation. Size selection was performed to obtain DNA fractions of 40-250bp ranges of MspI digested products. Afterwards, bisulfite treatment was conducted using ZYMO EZ DNA Methylation-Gold Kit™ following the manufacturer’s instructions. 12 cycles of PCR were performed to enrich the DNA fragments, in which each library was integrated with DNA index. The libraries were analyzed by Agilent 2100 Bioanalyzer and QPCR for quality control. Libraries were then subjected to paired-end 125bp multiplex sequencing on Hiseq 2500 platform.