title: GSM5497915: btl-GAL4::UAS-GFP – input (3); Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Chromatin from multiple independent collections of stage 11 – 16 btl-GAL4::UAS-rib-GFP and trh-GAL4::UAS-rib-GFP embryos were cross-linked at room temperature in 1.8% formaldehyde in 2 ml of homogenization buffer (60 mM KCl, 15 mM NaCl, 15 mM HEPES {pH7.6}, 4 mM MgCl2, 0.5 mM DTT, 0.5% Triton X-100 and cOmplete™ protease inhibitor cocktail [1 tablet per 50 ml buffer]). The cross-linked material was resuspended in 0.1% SDS and 0.5% N-lauroylsarcosine in lysis buffer (140mM NaCl, 15mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 1% Triton X-100, 0.5mM DTT and cOmplete™ protease inhibitor cocktail [1 tablet per 50 ml buffer]). Chromatin was sonicated three times at 4°C using the Sonic Dismembrator Model 100 (Fisher Scientific) under the following conditions: power setting 3, 20s ON, 20s OFF. Immediately after sonication, the chromatin extract was stored at −80°C prior to immunoprecipitation. Immunoprecipitations were performed, as described in (Negre et al., 2006), using a polyclonal goat anti-GFP antibody. DNA libraries were prepared using the NuGen Ovation Ultralow Library System (btl-GAL4::UAS-rib-GFP and trh-GAL4::UAS-rib-GFP experimental replicates 1 & 2 and corresponding input control), or the KAPA DNA HyperPrep Library Prep Kit (independent replicate of btl-GAL4::UAS-rib-GFP).  Illumina sequencing was performed according to the manufacturers specifications at the University of Minnesota Genomics Center (btl-GAL4::UAS-rib-GFP and trh-GAL4::UAS-rib-GFP experimental replicates 1 & 2 and corresponding input control) or at th UCLA Technology Center for Genomics & Bioinformatics (independent replicate of btl-GAL4::UAS-rib-GFP) as 50 bp single-end reads.