Reduced representation bisulfite sequencing of cadmium-exposed mouse placentas

The goal of the study was to identify regions of differential methylation between e18.5 mouse placentas exposed to cadmium and control placentas. Overall design: Animal work was approved by the North Carolina State University Institutional Animal Care and Use Committee, under protocols 16-045-B and 19-049-B. All mice were on a 14-hour/10-hour light/dark cycle at 30-70 % humidity, 22°C ± 4°C. 5-week-old C57Bl/6J (B) female mice were exposed for 5 weeks to 0, 1, or 50 ppm CdCl2 through drinking water (Sigma-Aldrich, 202908). At 10 weeks B mice were mated with unexposed CAST/EiJ (C) males. Cd exposure continued throughout mating and gestation. Embryos and placentae were dissected at e18.5; collected tissues were weighed, snap-frozen immediately upon dissection and stored at -80°C. Genomic DNA was isolated from four female 0 ppm and four female 50 ppm exposed placentae using the AllPrep DNA/RNA/miRNA kit (Qiagen). 100 ng of gDNA was digested with TaqaI (NEB) at 65°C for 2 hours followed by MspI (NEB) at 37°C overnight. Following enzymatic digestion, samples were used for library generation using the Ovation RRBS Methyl-Seq System (Tecan) following the manufacturer's instructions. In brief, digested DNA was randomly ligated, and, following fragment end repair, bisulfite converted using the EpiTect Fast DNA Bisulfite Kit (Qiagen) following the Qiagen protocol. After conversion and clean-up, samples were amplified resuming the Ovation RRBS Methyl-Seq System protocol for library amplification and purification. Libraries were measured using the Agilent 2200 TapeStation System and quantified using the KAPA Library Quant Kit ABI Prism qPCR Mix (Roche). Libraries were sequenced on a NextSeq 550 using single end 75 bp sequencing.