embedded into low-melt gel agarose plugs to prevent further shearing. Optimal digestions are selected by agarose pulsed field gel electrophoresis, with an optimal smear range from 1 MB to 20 to 100 KB, and are blunt-end ligated to a biotinylated linker. After secondary enzymatic digestion with MmeI, ligation of a second biotinylated linker and library amplification, the digested population is assayed using NGS [75]. In the Stamatoyannopoulos protocol, DNA from nuclei is digested with limiting DNase I concentrations and assessed by q-PCR and/or agarose gel electrophoresis. Optimal digestions are purified with size selection of fragments smaller than 500 bp using sucrose gradients, and are submitted for high-throughput sequencing after library construction [76]. The main difference between the two protocols is that the first one depends on the single enzymatic cleavage of chromatin, whereas the latter requires double cleavage events in close proximity to each other. The Stamatoyannopoulos protocol has been preferentially used by the ENCODE consortium.
