ChIP-Seq for Genome-Scale Analysis of Bacterial DNA-Binding Proteins

Abstract

Protein-DNA interactions are central to many basic biological processes, including transcription regulation, DNA replication, and DNA repair. Chromatin Immunoprecipitation (ChIP) is used to determine the position and strength of protein-DNA interactions in vivo. Coupling ChIP with microarrays (ChIP-chip), and more recently with deep sequencing (ChIP-seq), has allowed genome-wide profiling of DNA binding events in vivo. In this chapter we outline the steps to generate ChIP-seq libraries from bacterial samples and briefly discuss basic analysis of the data.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase

“…The cross-linking was quenched by the addition of 0.5 M glycine, and cells were harvested by centrifugation and washed once with tris-buffered saline. Cells were lysed as described by Bonocora and Wade ( 83 ) with lysis buffer containing lysozyme (4 mg/ml). Lysates were sonicated using the Bioruptor Pico (Diagenode) until most of the DNA fragments were between 300 and 500 bp.…”

“…Ribonuclease A was added to eliminate RNA-related interactions. The immunoprecipitation and library preparation were performed at the same time as described by Bonocora and Wade ( 83 ). Sequencing was performed on an Illumina MiSeq.…”

Dissectingpsalocus regulation inYersinia pestis

“…Briefly, KIM6+ and YPS24 ( psaE -His) were grown in HIB at 37 °C, pH6 to an OD 600 of 0.6 to 0.8. Sonicated lysates were prepared as previously described (Bonocora & Wade, 2015). ChIP-seq libraries were prepared as previously described (Bonocora & Wade, 2015), using 2 µg anti-His tag monoclonal antibody and 25 µL Protein G Sepharose slurry, or 2 µL M2 anti-FLAG antibody (Sigma) and 25 µL Protein A Sepharose slurry.…”

Defining bacterial regulons using ChIP-seq

“…Aspects of the ChIP-seq method and resulting data analysis will be described in detail throughout this review (Figure 1). Several methods and review articles describe in detail how to perform ChIP-chip and ChIP-seq experiments [30-33], and we encourage readers to also review these publications. The methodology described in these articles differs and while each has been used to produced high-quality ChIP-seq data, we have found success following the method described by Davis et al [33].…”

“…We have had success following the Illumina’s guidelines for library preparation, which is time intensive, involving many steps and reagents. A step-by-step protocol for ChIP-seq library construction is also described in a recent review [30]. Nevertheless, some samples may require additional troubleshooting during library preparation.…”