title: GSM6757752: OreR, GAF, nexus, 1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: For ChIP, 0.2-0.4 grams of fixed 2-3 h AEL embryos were used for all experiments. Chromatin extracts were prepared by douncing embryos in Lysis Buffer A1 (15 mM HEPES pH 7.5, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5% Triton X-100, 0.5 mM DTT (add fresh)), washing nuclei with ChIP Buffer A2 (15 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, and 0.1% SDS), and sonicating with a Bioruptor Pico (Diagenode) for six cycles of 30 seconds on and 30 seconds off. All ChIP-nexus experiments were performed using antibodies custom generated by Genscript: Zelda (aa 1117-1327), Dorsal (aa 39-346), Twist (C-terminus), Bicoid (C-terminus), Caudal (aa 1-214), GAF (aa 1-382). ChIP-seq experiments were performed with the following commercially available antibodies: H3K27ac (Active motif, 39133) and H3K4me1 (Active motif, 39635). ChIP-nexus was performed according to He et al., 2015, except that the ChIP-nexus adapter mix contained four fixed barcodes and PCR library amplification was performed directly after circularization of the purified DNA fragments (without addition of the oligo and BamHI digestion). ChIP-seq was performed as previously described in He et al., 2011, and included a whole cell extract (WCE). ChIP-nexus for transcription factors and ChIP-seq for histone modifications using standard Illumina protocols.