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).

本实验对既往报道的实验方案进行优化修改，开展染色质免疫共沉淀（Chromatin Immunoprecipitation, ChIP）操作（Lee等，2006；Lilja等，2007）。简言之，我们首先移除12小时龄胚胎的绒毛膜，以2%多聚甲醛进行交联固定，随后将胚胎重悬于储存缓冲液（50mM Tris-HCl、1mM EDTA，pH 8.0）中。将胚胎置于SDS裂解缓冲液（含1.0 mL 5 M NaCl、2.5 mL 1 M Tris-HCl pH 8.0、0.5 mL 0.5 M EDTA、2.5 mL 10% SDS）中进行裂解，之后重悬于含SDS与Triton的缓冲液（含1.0 mL 5 M NaCl、5.0 mL 1 M Tris-HCl pH 8.0、0.5 mL 0.5 M EDTA、2.5 mL Triton X-100及蛋白酶抑制剂混合物）中，于冰上进行超声破碎，使染色质片段的平均长度达到350 bp。将得到的剪切后染色质（25Izg）按照此前报道的方法（Lee等，2006），使用抗Myc抗体（Santa Cruz公司）进行免疫共沉淀反应。随后按照Illumina公司的试剂盒说明书构建ChIP测序（ChIP-sequencing）文库，最终获得的DNA文库采用Illumina测序平台完成测序，测序工作由马萨诸塞大学医学院核心实验室完成。