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. Chromatin (0.5 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) was diluted 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).