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.