ChIP-Seq of constitutively expressed Ndt80-Myc in S. cerevisiae

The purpose of this experiment was to identify the genes bound by Ndt80 in S. cerevisiae when constitutively expressed, for the native Ndt80 as well as a heterologous Ndt80 from Pichia pastoris. For the ChIPs on the native Ndt80s, Ndt80 was tagged with c-myc; for the heterologous ChIP P. pastoris Ndt80 was integrated into the S. cerevisiae genome and tagged with c-myc. In both cases, the native Ndt80 promoter was also replaced with an A. gossypii Gal1 promoter and the protein was immunoprecipitated with a c-myc antibody. Cells were grown overnight in SRaffinose until log-phase, then in SRaffinose + 1.7% Galactose for 5 hours. Each experiment was repeated twice and sequenced on an Illumina HiSeq 4000.本实验旨在鉴定组成型表达状态下，酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）中与Ndt80结合的靶基因，实验对象涵盖内源Ndt80以及来自毕赤酵母（Pichia pastoris）的异源Ndt80。针对内源Ndt80的染色质免疫共沉淀（Chromatin Immunoprecipitation，ChIP）实验中，Ndt80被标记有c-myc标签；而异源Ndt80的ChIP实验则将毕赤酵母Ndt80整合至酿酒酵母基因组中，同样为其添加c-myc标签。两组实验均将内源Ndt80的启动子替换为棉阿舒囊霉（Ashbya gossypii，A. gossypii）的Gal1启动子，并使用c-myc抗体对目标蛋白进行免疫沉淀。细胞先在棉子糖培养基中过夜培养至对数生长期，随后转移至添加1.7%半乳糖的棉子糖培养基中继续培养5小时。每项实验均重复两次，并采用Illumina HiSeq 4000测序平台完成测序。