title: GSM8967679: liver924_ControlBPA; Ovis aries; Bisulfite-Seq
library_name: GSM8967679
library_construction_protocol: At the conclusion of the second breeding season (~21 months of age), female offspring in the T cohort were subjected to ovariectomy to eliminate any confounding influences from varying steroid levels. Liver tissue samples were obtained during the artificially induced follicular phase, following established protocols. Early follicular phase estradiol levels were maintained using a subcutaneous estradiol implant, 1 cm in length. To synchronize estrous cycle, two controlled internal drug-release implants containing progesterone (CIDR-G; InterAg, Hamilton, New Zealand) were placed subcutaneously to mimic luteal phase levels of progesterone. The progesterone implants were removed after 14 days, and four 30-mm estradiol implants were inserted to produce late follicular phase levels of estradiol. Animals were euthanized 18 hours later during the late artificial follicular phase by a barbiturate overdose (Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI). In the prenatal BPA-treated cohort, female offspring liver tissue samples were collected during the natural follicular phase after synchronization with two injections of Prostaglandin F2alpha, 11 days apart. Samples were taken 27 hours after the second Prostaglandin F2alpha injection. From all animals, liver tissue was collected from the tip of the left lobe, flash-frozen and stored at -80°C. DNA was extracted from 30 micrograms of frozen liver tissue using AllPrep DNA/RNA kit (Qiagen, Germantown, MD) using manufacturer protocol. A total of 200ng of DNA was used for each whole genome bisulphite sequencing (WGBS).  Library preparation and WGBS were carried out at the University of Michigan, Epigenomics Core. Each sample was spiked-in with 0.5% (w/w) of unmethylated Lambda DNA prior to library preparation, according to the ENCODE consortium's guidelines. DNA was fragmented to a 350bp peak on a Covaris S220 focused-ultrasonicator system. The samples were next processed for end-repair and A-tailing, before proceeding to the ligation of a truncated methylated adaptor at 16°C overnight. The ligation products were cleaned with two rounds of Omega Bio-tek Mag-Bind TotalPure NGS beads. The ligated products were submitted to bisulfite treatment using Zymo's EZ DNA Methylation kit, with the following protocol: 55 cycles of 95 degrees C for 30sec, 55 degrees C for 15 minutes. Samples were kept at 4 degrees C until cleanup. Final library amplification was done using the KAPA HiFi Uracil+ polymerase and NEB's dual indexing primers. A total of 10 cycles of amplification were used. The PCR products were then cleaned with Omega Bio-tek Mag-Bind TotalPure NGS beads, and the final libraries were quantitated using the Qubit High Sensitivity dsDNA kit. Libraries were quantified using the KAPA Library quantitation kit and pooled for sequencing with a S4 flowcell for 200 cycles (paired end, 100 base pairs in length) on a NovaSeq6000.