Context dependent function of transcriptional regulator Rap1 in gene silencing and activation [ChIP-seq timecourse]

As part of a study on the function of Rap1 at HML and MAT in Saccharomyces cerevisiae, we performed an Anchor Away experiment on Rap1, inducibly depleting it from the nucleus upon addition of Rapamycin, followed by a ChIP-seq time-course. This was done in a strain that had SNPs introduced to HMLalpha to make it distinguishable from MATalpha. Samples were spiked with a constant amount (5% by OD) of a S.paradoxus strain that also expressed 2xV5-Rap1. Reads from the S.paradoxus sample served as the normalization factor. Peaks were then fit to a non-linear regression model to extract the k(off) and apparent residence time in vivo.本研究围绕酿酒酵母（Saccharomyces cerevisiae）中Rap1在HML与MAT位点的功能展开，针对Rap1开展锚定撤离（Anchor Away）实验：加入雷帕霉素（Rapamycin）后可诱导Rap1从细胞核内耗竭，随后进行染色质免疫沉淀测序（ChIP-seq）时间进程实验。本次实验所用菌株经基因编辑，在HMLalpha位点引入单核苷酸多态性（SNP），使其序列与MATalpha得以区分；实验样本以光密度（OD）占比5%的恒定剂量，掺入同时表达2xV5-Rap1的奇异酵母（S.paradoxus）菌株，奇异酵母样本的测序读段被用作标准化因子。最终将染色质结合峰拟合至非线性回归模型，以提取体内解离速率常数（k(off)）与表观驻留时间。