title: GSM6456231: gd7 2to4h ChIP CBP Replicate 2; Drosophila melanogaster; ChIP-Seq
library_name: GSM6456231
library_construction_protocol: To prepare chromatin, embryos were homogenized in a glass dounce homogenizer by 20 strokes with a tight pestle in A1 buffer (15 mM HEPES pH 7.6, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5 mM DTT, 0.5% Triton X-100 and protease inhibitor tablets (Roche)), centrifuged at 3500 g for 5 min at 4°C and the supernatant discarded. The remaining nuclear pellet was resuspended in 200 μl of sonication buffer (15 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% sodium deoxycholate, 1% Triton X-100 and protease inhibitor tablets (Roche)) supplemented with 0.5% SDS and 0.2% n-lauroylsarcosine and sonicated using a Bioruptor (Diagenode) with high power settings to obtain an average fragment size distribution of 200-500 bp, Sonicated chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and diluted 5-fold in sonication buffer to reduce the concentration of detergents. Immunoprecipitations (IPs) were performed with 10 μg rabbit anti-CBP (homemade) Chromatin was incubated with anti-CBP overnight at 4°C and equal amounts of Protein A and G Dynabeads (Invitrogen), pre-blocked with BSA (1 mg/ml), were incubated with the samples for 4 h at 4°C. Chromatin corresponding to 10% of the amount in each IP was withdrawn to serve as an input for qPCR. Samples were subjected to 10 min washes with Wash A (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 0.1% SDS, 0.1% Sodium Deoxycholate and 1% Triton X-100), Wash B (Wash A adjusted to 500 mM NaCl), Wash C (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% Sodium Deoxycholate and 0.5% IGEPAL CA-630) and Tris-EDTA (TE) buffer. Beads were resuspended in 100 μl TE and treated with RNase A (20 μg/ml) at 55°C for 30 min before SDS (to 0.75%) and Tris-HCl (to 50mM) were added and crosslinks reversed at 65°C for overnight. Eluted ChIP DNA was treated with Proteinase K at 55°C for 2 h and purified with the ChIP DNA Clean & Concentrator kit (ZymoResearch, D5205). ChIP-sequencing was performed using 2-5 ng of ChIP DNA in biological duplicates. Libraries were prepared with the NEBNext® Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and single-end (1 x 75 bp) sequenced on the Illumina NextSeq 550 platform at the BEA core facility, Karolinska Institutet, Stockholm.