Schematic illustration of procedures for the three methodologies used to survey embryo methylome and hydroxymethylome. A) Me-RDA and HMe-RDA methodologies use hybridization and subtraction between Tester DNA cleaved with an insensitive enzyme and Driver DNA cleaved with a sensitive isoschizomer. White circle: unmethylated cytosine; orange circle: methylated cytosine. 1 The subtractive PCR includes a single-strand DNA digestion step by a mung bean nuclease after 10 cycles of amplification and is followed by 20 cycles of amplification. B) HELP Cocktail methodology. Following TasI ligation mediated-PCR, amplification products are digested with a methyl sensitive enzymatic cocktail. Methylated fragments remain uncut, and are subject to exponential amplification in a final PCR.