Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation

Abstract

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein-DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

Keywords: Brn1; Chromatin immunoprecipitation; Cohesin; Condensin; Meiosis; Mitosis; Rec8; Saccharomyces cerevisiae; Scc1; Schizosaccharomyces pombe.

Figure 1

Schematic overview of the workflow. Yeast cells are cross-linked to stabilize DNA–protein interactions. Cells are subsequently lysed, and chromatin then sheared. Proteins of interest are immunoprecipitated using protein-specific antibodies immobilised on Protein G Dynabeads. Samples are either analysed by quantitative real-time PCR or DNA is further processed for ChIP-seq library preparation and subsequent deep sequencing.

Figure 2

A. Representative image of mitotic yeast cells sonicated with a Bioruptor Twin (Diagenode) for a 30-minute round (power setting: High, 30 s ON / 30 s OFF). DNA from two different samples was loaded on a 2% agarose gel with a 100 bp marker ladder. B. Representative optimal BioAnalyzer trace (upper panel) and contaminated trace with adapter (bottom panel) C. Examples of profiles generated by chromatin immunoprecipitation followed by sequencing (ChIP–seq) of the cohesin subunit Scc1 in wild type cells (IP shown in blue; Input shown in grey) and calibrated with S. pombe Scc1 distribution in representative chromosome V (IP shown in blue, bottom panel).