Embryos were collected for one hour and aged appropriately, then dechorionated and dounce homogenized. The homogenate was passed through Miracloth, further homogenized and nuclei were pelleted. Alternatively, Kc167 cells were cultured in Schneider's medium supplemented with 10% FBS. Cells were centrifuged, resuspended in 0.025% IGEPAL for 5.5 minutes and nuclei were pelleted.

Nuclei from embryos or Kc167 cells were treated with DNaseI as previously described (PMID:16791208). Samples were treated with Proteinase K, phenol:chloroform extracted and fractionated through a sucrose gradient to isolate fragments in the 100-400 bp range. These fragments were used to generate a library, single-end layout.

DNaseI cleavage sites were characterized by Illumina GA1 sequencing.

An average of 13.4 million DNAse I cleavage events per sample were mapped to D. melanogaster genomic sequence. To identify regions of enriched accessibility, the number of tags within a 250-bp scanning window was compared to the expected number of tags based on a binomial model of the surrounding 50 kb to determine an enrichment z-score. Accessible regions were defined as collections of adjacent tags with z-scores greater than T where the number of background (random) regions with z ≥ T represent 1% of the number of experimental regions with z ≥T (that is, a 1% FDR control). DNAse I tag density genome-wide was calculated by dividing the genome into 20-bp bins and adding the number of tags within a 150-bp window around each bin. The density scores were then used to identify peaks in accessibility within accessible regions, with each 150-bp peak being designated a DHS. The peak detection method allowed multiple DHSs per accessible region.