GSM7061116: Tet-GFP, 0-12h Embryo, Rep2; Drosophila melanogaster; ChIP-Seq
0-12h embryos were collected, processed, and chromatin was prepared according to Yad et al. 29, except lysates were sonicated on a Covaris S2 sonication device (intensity 8, duty cycle 20%, cycle burst 200) for 30 minutes at 4˚C to reach fragments ranging from 150–500 bp and then centrifuged at 20,000g at 4˚C for 1 minute. Supernatants were collected and centrifuged again for 15 minute to remove debris. Chromatin samples were then snap frozen in dry ice and stored at -80˚C until immunoprecipitation in triplicets. All buffers contained cOmplete EDTA-free protease inhibitor cocktail (Roche). NGS Libraries were made from eluted DNA using the NEBNext Ultra II DNA Library Prep kit (New England Biolabs) according to the manufacturer's protocol. Briefly, 20 ng of DNA fragments were end-repaired and the blunt, phosphorylated ends were treated with Klenow DNA polymerase and dATP to yield a 3′ A base overhang for ligation of Illumina adapters. After adapter ligation, DNA was PCR amplified with indexed primer for 12 cycles. Libraries were size-selected using Ampure XP beads (Beckman Coulter) to remove adapter dimers. DNA was quantified by fluorometry with the Qubit 2.0 (Thermo Scientific) and DNA integrity was assessed with a Fragment Analyzer (Agilent). The libraries were pooled and sequenced on the NextSeq 500 platform using 75 bp single end sequencing according to manufacturer's protocol using Reagent v.2.5 at the Waksman Institute Genomics Core.