title: GSM2616874: nonstop_ip_1924_1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Embryos were fixed in 1.8% formaldehyde for 15 min at room temperature. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM. Embryos were homogenized in buffer A1 (15 mM HEPES at pH 7.5, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5% TritonX-100, 0.5 mM DTT) (0.1g embryos/1ml A1). The chromatin pellet was washed three times with 5ml buffer A1 and once with 5ml buffer A2 (15 mM HEPES at pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritonX-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroyl sarcosine). The chromatin was sonicated in 250μL of buffer A2 (0.1g embryos/250ul A2) seven times for 10 sec (0.9s on/0.1s off for 10 times) at 30% power. After centrifugation at 14,000 rpm for 10 min at 4°C, 250 μL of clarified soluble chromatin was used for ChIP as described previously (Huang et al. 2014). The antibodies used for ChIP included 10 μg of rabbit α-Sgf11, 20 μg of rabbit α-Ada2b, 20 μg of rabbit α-Spt3, 20 μg of rabbit α-Non-stop, 3 μg mouse α-pol2(4H8) (ab5408, Abcam). For ChIP-seq library preparation, 10ng ChIP DNA and WCE DNA were used with the KAPA HTP Library Prep Kit for Illumina and Bioo Scientific NEXTflex DNA barcodes. The resulting libraries were purified using the Agencourt AMPure XP system (Beckman Coulter) then quantified using a Bioanalyzer (Agilent Technologies) and a Qubit fluorometer (Life Technologies).  Post amplification size selection was performed on all libraries using a Pippin Prep (Sage Science).  Libraries were re-quantified, normalized, pooled and sequenced on an Illumina HiSeq 2500 instrument as 50-bp single read.