Pharate pupal stage was confirmed by darkening of wing and bristles.
Staged tissue was fixed using 1% formaldehyde for 10 minutes at room temperature. Tissue was then homogenized and filtered through a 60 micron nitex membrane. Nuclei were pelleted at 4500g for 20 minutes then resuspended and disrupted using bead-beating with 2mm tungsten beads. Chromatin was then sonicated to a size range of 500 bp to 2 kb. Soluble chromatin was recovered after centrifugation at 15000g for 5 minutes.
Soluble chromatin was phenol-chloroform extracted. DNA was isolated from the aqueous phase.
Isolated DNA was characterized by high-throughput sequencing using the "Illumina HiSeq2000" platform.
