Chromatin was extracted from 50 µl aliquots of frozen wildtype fly heads, aged between 0 and 5 days old, in biological duplicates. Fly heads were crushed in PBS (Sigma) and crosslinked with formaldehyde (Sigma) at a final concentration of 1% for 30 minutes. Crosslinking was terminated using glycine (Invitrogen) at a final concentration of 125mM and crosslinked material was immediately washed twice in PBS and centrifuged at 13000rpm, for 15 minutes at 4 degrees. The pellet was resuspended in buffer 1 (15 mM Tris-Hcl (pH 7.5), 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.1 mM sucrose) and the solution was homogenized using a QiaShredder column (Qiagen). Cells were lysed using buffer 1 supplemented by 2% triton-X-100 (Sigma) and a crude nuclear extract was collected by centrifugation (6000 rpm, 10 minutes at 4 degrees). Nuclei were re-suspended in incubation buffer (0.15% SDS, 1% triton x-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris) supplemented with 0.1% BSA (Sigma) and 1x protease inhibitor (Roche) and subjected to sonication (Diagenode Bioruptor) for 30 minutes (30 seconds on/off cycle using the “high intensity” mode), yielding average DNA fragments of 150-300 base pairs. Immunoprecipitation reactions were performed overnight in incubation buffer with 3 ng of anti-trr antibody (gift from Dr. A. Mazo), protease inhibitor cocktail (Roche), BSA, and pre-blocked protein A/G agarose beads (Santa Cruz). Chromatin-antibody-bead complexes were recovered by centrifugation (4000rpm, 2 minutes at four degrees) and washed twice with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris pH 8, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris pH 8, 500 mM NaCl), once with LiCl wash buffer (10 mM Tris pH 8.0, 1% Na-deoxycholate, 1% NP-40, 250 mM LiCl, 1 mM EDTA) and twice with TE buffer. Chromatin was eluted in 1% SDS, 0.1M NaHCO3, 200 mM NaCl and decroslinked at 65° Celcius (C) for four hours. DNA was purified by phenol/chloroform extraction and ethanol precipitated using linear acrylamide (Ambion) and sodium acetate at -20 degrees. Library preparation for Illumina sequencing was performed using the Truseq DNA sample prep kit V2 (Illumina) with approximately 3 ng of starting DNA 15 PCR cycles for amplification. Library fragment size was assessed using the 2100 Agilent Bioanalyser and was shown to be between 200 and 400 bp.
Sequenced DNA Library
ChIP-seq libraries were prepared for sequencing using standard Illumina protocols (Trueseq)