Traditionally, identification of DHSs has been based on Southern blotting with indirect end-labeling [28] and involves laborious and time-consuming steps that limit the applicability of the method to a narrow extent of the genome. Further attempts to improve the efficiency and resolution of the method have used low-throughput sequencing, real-time PCR strategies and later hybridization to tiled microarrays [68–74]. The advent of NGS gave rise to DNase-seq allowing the genome-wide identification of DHSs with unparalleled specificity, throughput and sensitivity in a single reaction. In recent times the drop of sequencing costs and the increased quality of the data have made DNase-seq the ‘golden standard’, for probing chromatin accessibility. During a typical DNase-seq experiment, isolated nuclei are submitted to mild DNase I digestion according to the Crawford or Stamatoyannopoulos protocol [75, 76]. In the Crawford protocol, DNase I digested DNA is 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
