Genome-Wide Quantification of Protein-DNA Interactions in Chlamydia Using ChIP-Seq (Chromatin...

Chromatin immunoprecipitation (ChIP) measures binding between a specific protein and its in vivo DNA-binding sites. It is challenging to perform ChIP for bacteria that reside inside a eukaryotic cell, but we have optimized this method for Chlamydia-infected cells and used it to identify genome-wide binding sites of chlamydial transcription factors and sigma factors. The ability to measure protein-DNA interactions in intracellular chlamydiae without the need to isolate bacteria from the host cell allows changes in DNA binding to be measured during the Chlamydia developmental cycle and under different growth conditions. In this chapter, we provide a detailed protocol for Chlamydia ChIP by describing how to cross-link proteins to DNA, shear chlamydial chromatin into DNA fragments, and immunoprecipitate protein-DNA complexes with specific antibodies against the protein of interest. After a decrosslinking step, DNA fragments are analyzed by qPCR to measure binding to individual DNA-binding sites, or with deep sequencing for unbiased identification of genome-wide binding sites.