ChIP-seq data processing and relative and quantitative signal normalization for Saccharomyces cerevisiae
Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) is a widely used technique for genome-wide analyses of protein-DNA interactions. This protocol provides a guide to ChIP-seq data processing in Saccharomyces cerevisiae, with a focus on signal normalization to address data biases and enable meaningful comparisons within and between samples. Designed for researchers with minimal bioinformatics experience, it includes practical overviews and refers to scripting examples for key tasks, such as configuring computational environments, trimming and aligning reads, processing alignments, and visualizing signals. This protocol employs the sans-spike-in method for quantitative ChIP-seq (siQ-ChIP) and normalized coverage for absolute and relative comparisons of ChIP-seq data, respectively. While spike-in normalization, which is  semiquantitative, is addressed for context, siQ-ChIP and normalized coverage are recommended as mathematically rigorous and reliable alternatives. Overall design: S. cerevisiae ChIP-seq profiling of Hho1 and Hmo1 was performed in two independently derived strains (biological replicates; Hho1: yTT6336 and yTT6337; Hmo1: yTT7750 and yTT7751), with samples collected at three distinct cell cycle stages: G1, G2/M, and quiescence (Q). The HHO1 and HMO1 genes were separately tagged with a 3xFLAG epitope, including flexible linkers (2L-3FLAG), generating HHO1-2L-3FLAG and HMO1-2L-3FLAG strains. As there is no indication that the tag compromises the function of either Hho1 or Hmo1, these strains are labeled "WT" (wild type) in file names. Spike-in chromatin from Schizosaccharomyces pombe was incorporated into the immunoprecipitation reactions to facilitate normalization; the S. pombe spike-in strain (Sphc821) contains abp1-3FLAG.