A total of 9 embryos (control, alcohol with NTC phenotype, and Alcohol with NTO phenotype, n = 3 each) were used for MeDIP analysis. Genomic DNA was extracted from frozen mouse control and alcohol-treated embryos (neural tube open vs. neural tube closed phenotypes) in triplicate using a DNeasy Tissue Kit (Qiagen, Fremont, CA). Briefly, embryos were dounced using a homogenizer on ice and lysed with Proteinase K and tissue lysis buffer for 3 hrs, then precipitated, washed and eluted in elution buffer according to the manufacturer’s instructions. The DNA quality and quantity was assessed using a Nanodrop spectrophotometer with A260 ratio >1.7 and A230 >1.6 considered to be a criteria for quality control. Approximately, 6 µg of genomic DNA from each embryo were sonicated using a Branson sonifier to obtain fragment sizes between 200–1,000 bp (verified on 2% agarose gel; Suppl. Fig. S4). The sonicated DNA, diluted in TE buffer, was heat-denatured for 10 min at 95°C. 20 µl of DNA was removed and stored at −20°C and used as the input DNA sample. The remaining samples were incubated overnight
