Bicoid binding along anteroposterior axis of Drosophila Melanogaster embryos

ChIP-Seq against Bicoid on manually dissected posterior thirds of Drosophila Melanogaster early stage 5 embryos. Overall design: Embryos were collected from a population cage for 90 minutes and then aged for 2 hours in order to enrich for embryos at developmental stage five. Embryos were then fixed with formaldehyde as previously described (Li et al. 2008) and sorted by morphology for those at early stage 5. The posterior thirds of embryos were sliced off by hand with a scalpel. A pool of the embryo segments from approximately 300 embryos was combined with 20 µg of whole Drosophila pseudoobscura embryos at stage 5 (to serve as carrier), and homogenized in homogenization buffer containing 15mM NaCl, 15mM TrisHCl pH 7.5, 60mM KCl, 1mM EDTA, and 0.1% Triton-X100, with 1mM DTT, 0.1 mM PMSF, and protease inhibit cocktail (Roche) added before use. After homogenization, 0.5% NP40 was added, and following a 5 minute incubation, samples were spun down at a low speed. Nuclei in the pellet were then washed once with the homogenization buffer containing 0.2M NaCl. The low speed centrifugation was repeated, and the recovered nuclei pellet was then re-suspended in nuclear lysis buffer (10 mM TrisCl, pH 7.9, 100 mM NaCl, 1 mM EDTA, 0.5 % Sarkosyl) +1%SDS, +1.5% sarkosyl. The chromatin was recovered by spinning the sample at full speed in a micro-centrifuge at 4oC for 1 hour and was re-suspended in a small volume of nuclear lysis buffer. Chromatin was sheared to an average size of 300bp using a Covaris sonicator (peak power= 140, duty factor = wo, cycle burst = 200, time = 2:20 minutes). Chromatin immunoprecipitation was performed using 72ng of chromatin and 1.5ug of an anti-Bicoid polyclonal antibody described previously (Li et al. 2008). The Bicoid antibody was coupled to Dynabead M-280 sheep anti-rabbit magnetic beads and the immunoprecipitation was conducted with the standard protocol from the manufacturer. DNA libraries for the chromatin immunoprecitation samples were prepared using the Rubicon genomics thru-plex DNA-seq kit using 16 PCR cycles and sequenced using Illumina High-seq with 2500 rapid run100bp single end reads. The sequencing reads were aligned to a combined D. pseudoobscura (Flybase Release 1.0) and D. melanogaster genome (Flybase Release 5) using Bowtie2 with options set as bowtie2 -5 5 -3 5 -N 1 -X 2000. The aligned reads were converted to wig files using custom scripts available upon request. Wig files were normalized to 10 million mapped reads.