DNA isolation and methylation assay

Genomic DNA was isolated as previously described [60] from three placentas per dam, chosen by using a random number generator. Briefly, tissues were lysed in lysis buffer with proteinase K followed by separation using Phenol:Chloroform:Isoamyl Alcohol. Genomic DNA samples were stored at -20⁰C until use. DNA concentration was measured using an Epoch Microplate Spectrophotometer (Bio-Tek Instruments).

The level of global DNA methylation was measured using the 5-mC DNA ELISA kit (Zymo Research) according to the manufacturer's protocol, with the following exceptions: (1) Antibody incubation was performed overnight at 4⁰C; (2) 200ng of DNA was used per standard and sample well. To construct a standard curve, methylated DNA (Zymo Research) and unmethylated Lambda DNA (New England BioLabs) were mixed in various proportions (12.5%, 6.25%, 3.125%, 1.56%, 0.78%, and 0% methylated DNA). Intra-assay coefficient of variation was 5.41%. Samples were run in duplicate. To calculate the percentage of CpG that are methylated, the %5-methylcytosine (mC) calculated from the standard curve was corrected for the differences in average CpG density between mouse and E.Coli (used for the standard curve) genomic DNA, according to manufacturer’s instructions.