trr_ChIPseq_wildtype_rep2
Drosophila melanogaster
source_name
Drosophila heads
tissue
head
developmental stage
adult (0-5 days old)
genotype
Wild type
chip antibody
anti_Trr (Johnston et al., 2011)
extraction protocol
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.