Schematic diagram showing the strategy of mapping hypomethylated sequences in the genome by TspRI–HpaII–ExoIII digestion. Genomic DNA was first digested with TspRI, which created DNA fragments with a nine-base 3′ extension and were resistant for exonuclease III digestion. The DNA fragments were then digested with methylation-sensitive restriction enzyme HpaII. After treatment with exonuclease III and RecJf exonuclease, ExoIII-digested or mock-digested DNA was purified and labeled with Cy3 and Cy5 fluorescence dyes, respectively. Cy3 hybridization intensity was normalized to Cy5 for comparison among samples.