title: GSM5268391: C646 dRING Rep 2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Cells were fixed in 1% formaldehyde (Sigma Aldrich, F8775) for 15 min at room temperature, quenched with 0.16 M glycine for 5 min at 4°C and washed in cold PBS. Crude nuclear extracts were obtained by sequentially washes with ChIP buffer A (10 mM HEPES pH 7.6, 10 mM EDTA pH 8.0, 0.5mM EGTA pH 8.0 and 0.25% Triton X-100) followed by ChIP buffer B (10mM HEPES pH 7.6, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 and 0.01% Triton X-100) to isolate nuclei. Nuclei were resuspended in 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 and 1% Triton X-100), with Complete Mini EDTA-Free protease inhibitor tablets, Roche) supplemented with 0.5% SDS and 0.2% n-lauroylsarcosine, at a final concentration of 5 – 10 x 107 cells/ml and sonicated in a Bioruptor (Diagenode) using high power settings to obtain an average fragment size distribution of 200–500 bp. The concentration of chromatin was quantified and spike-in chromatin from Drosophila virilis cells was added. Immunoprecipitations were performed by incubating 10 μg of chromatin with anti-CBP (homemade), anti-dRING (kind gift of Yuri Schwartz, Umeå Universitet, Sweden), anti-E(z) (kind gift of Richard Jones, Southern Methodist University, USA) and anti-Pho (kind gift of Judith Kassis, National Institute of Health, USA) overnight. Chromatin-antibody complexes were bound to Protein A and G Dynabeads (Invitrogen) and washed sequentially with Wash A (10 mM Tris 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 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). Beads were resuspended in 100 μl TE, treated with RNase A (20 μg/ml) at 55°C for 30 min, supplemented with SDS (0.75%) and Tris (50 mM). and crosslinks reversed at 65°C overnight. Eluted ChIP DNA was treated with Proteinase K at 55°C for 2 h and purified using the ChIP DNA Clean & Concentrator kit (ZymoResearch, D5205). Libraries were constructed from ChIP samples (2-5 ng) with the NEBNext Ultra II DNA Library Prep Kit (NEB) and single-end (1x75 bp) sequenced on an Illumina NextSeq 550 platform at the BEA core facility, Stockholm.