Identification of E93 binding sites in Drosophila melanogaster

Chromatin immunoprecipitation was performed with modification of the protocols described before (Lee et al., 2006; Lilja et al., 2007). Briefly, the chorion was removed from 12-hour old embryos, cross-linked using 2% paraformaldehyde and resuspended in storage buffer (50mM Tris-HCl pH8.0, 1 mM EDTA). Embryos were then lysed in SDS-lysis buffer (1.0 ml 5 M NaCl, 2.5 ml 1 M Tris-HCl pH 8.0, 0.5 ml 0.5M EDTA, 2.5 ml 10 % SDS), resuspended in SDS-lysis and Triton buffer (1.0 ml 5 M NaCl, 5.0 ml 1 M Tris-HCl pH 8.0, 0.5 ml 0.5M EDTA, 2.5 ml Triton X-100, protease inhibitor cocktail) and sonicated on ice to an average length of 350 bp. The resulting sheared chromatin (25Izg) was subjected to immunoprecipitation using anti-Myc antibody (Santa Cruz) as described previously (Lee et al., 2006). ChIP-sequencing libraries were constructed following manufactureras instructions (Illumina). The resulting DNA libraries were sequenced using Illumina platform (University of Massachusetts Medical School Core Facility).