Illumina HiSeq 2000 sequencing; Opbp ChIP-seq in Drosophila melanogaster embryos
Opbp ChIP-seq in Drosophila melanogaster embryos
Wildtype Drosophila melanogaster (Oregon R) flies were grown in population cages at 25 degrees. Three rounds of 0-1hr prelays were performed to stimulate egg release from the females. Embryos were collected for 12 hours. The collected embryos were dechorionated using 50% bleach and fixed in 10 ml cross-linking solution (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) with 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collection were set aside for staging. Approximately 1g of Drosophila embryos were resuspended in 15ml ice-cold 0.1% Triton X-100 in PBS with protease inhibitors (PIs) and dounced 20x with loose pestle on ice, followed by centrifugation at 400g for 1min at 4 degrs. The supernatant was transferred to a fresh tube and centrifuged at 1100g for 10min at 4 degrs. The pellet was resuspended in 15ml ice-cold Cell lysis buffer with PIs (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP40) and dounced 20x with tight pestle. Two equal aliquots were transferred into 15ml falcon tubes and centrifuged at 2000g for 4min at 4 degrs. The pellets consisting of nuclei were resuspended in 1ml of ice-cold Nuclear Lysis Buffer with PIs (50 mM HEPES pH 8.0, 10 mM EDTA, 0.5% N-laurylsarcosine) and incubated for 20min at RT. 1ml ice-cold Nuclear Lysis Buffer with PIs was added to each tube and sonicated using the bioruptor for 15 cycles (15s on/ 15s off, at 4 degrs). Chromatin was then transferred to 1.5 ml-eppendorf tubes and centrifuged at 14000 rpm for 10 min at 4 degrs. Finally, supernatants were pooled, aliquoted and frozen in liquid nitrogen. Chromatin (50 ug of DNA) was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF) and incubated with anti-Opbp antibody at 4 degrs on a rotary shaker overnight. The antibody/antigen complexes were precipitated with 40 ul protein G sepharose at 4 degrs for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with 500 mM NaCl. Sepharose was resuspended in TE with 0.5% SDS, treated with RNase A for 30 min and protease K over night, incubated at 65 degr for 6 hours and phenol chloroform extracted. Solexa libraries were prepared according to manufacturers recommendations with small modifications. In short, 1-10ng of purified, RNase treated, and reverse cross-linked genomic DNA was end-repaired and terminal adenosine residues were added using the NEBNext reagents. Custom-made indexed adapters were ligated, after which the material was size selected at ~200-600 bp with Ampure XP beads (Beckman Coulter). PCR amplification was performed using PE1.0 and PE2.0 primers (custom-made) for 12 cycles for Input samples and 14 to 15 cycles for IP-ed samples using the Q5 Hot Start HiFi PCR Master Mix (NEB). The PCR-amplified library was purified using Ampure XP beads and its quality was assessed on a Bioanalyzer 2100 system (Agilent).