Chromatin immunoprecipitation (ChIP) measures the physical association between a protein and DNA in the cell. In combination with next-generation sequencing, the technique enables the identification of DNA targets for the corresponding protein across an entire genome. Here we describe the immunoprecipitation of Vibrio cholerae DNA bound to the histone-like nucleoid structuring protein (H-NS) tagged with the Flag epitope. The quality of the DNA obtained in this protocol is suitable for next-generation sequencing. The procedure described herein can be readily adapted to other bacteria and DNA-binding proteins.
Key wordsTranscription DNA-binding proteins Immunoprecipitation Chromatin immunoprecipitation Next-generation sequencing Vibrio cholerae
This work was supported by Public Service Research Grants and a Ruth L. Kirschstein National Research Service Award from the National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, USA.
- 6.Wang H, Ayala JC, Benitez JA et al (2012) Interaction of the histone-like nucleoid structuring protein and the general stress response regulator RpoS at Vibrio cholerae promoters that regulate motility and hemagglutinin/protease expression. J Bacteriol 194:1205–1215CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Wang H, Ayala JC, Benitez JA et al (2015) RNA-Seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis. PLoS One 10:e0118295. https://doi.org/10.1371/journal.pone.0118295 CrossRefPubMedPubMedCentralGoogle Scholar