Chromatin Immunoprecipitation of Meiotically Expressed Proteins from Arabidopsis thaliana Flowers

Abstract

During meiosis recombination occurs between homologous chromosomes which can result in reciprocal exchanges of genetic information, called crossovers. Crossover rate is heterogeneous within the genome, with local regions having a significantly higher recombination rate relative to the genome average. These regions are termed hotspots and typically occur with widths of kilobases. Therefore, there is a need to profile recombination factors at a similar resolution during meiosis via techniques such as chromatin immunoprecipitation (ChIP). Here we describe a ChIP protocol, combined with high throughput sequencing (ChIP-seq) optimised for analysis of meiotically expressed proteins in Arabidopsis thaliana flowers. We provide methods to (1) isolate nuclei and prepare the chromatin for shearing, (2) immunoprecipitate DNA molecules cross-linked to a protein of interest, (3) to size-select and purify immunoprecipitated DNA molecules, and (4) to prepare DNA sequencing libraries suitable for high-throughput sequencing. Together, these methods allow the detection of binding sites for meiotic proteins in the Arabidopsis genome at high resolution, which will provide insights into relationships between meiotic chromosome organization, chromatin and recombination.