ChIP-seq analysis of genome-wide DNA binding by transcription factors HapR and LuxO in Vibrio cholerae
ChIP-seq analysis of genome-wide DNA binding by transcription factors HapR and LuxO in Vibrio cholerae
The Vibrio cholerae O1 El Tor strain E7946 was isolated in Brazil (Levine MM, Black RE, Clements ML, Cisneros L, Saah A, Nalin DR, et al. The pathogenicity of nonenterotoxigenic Vibrio cholerae serogroup O1 biotype El Tor isolated from sewage water in Brazil. J Infect Dis. 1982;145: 296–299). Cells were grown in liquid LB minimal medium at 37 degrees with shaking. Immunoprecipitations were done as described by Haycocks et al. (PLoS Pathog. 2015. 11:e1004605) using Vibrio cholerae strain E7946. After growing cells to midlog phase, crosslinking, and sonication, protein DNA complexes were immunoprecipitated using anti-FLAG.   Libraries were prepared using immunoprecipited protein-DNA complexes immobilised with Protein A-sepharose. DNA fragments were then blunt ended, A-tailed and barcoded. This was done using an NEB quick blunting and ligation kit, the Klenow fragment (5'-3' exo-, NEB) and NEXTflex ChIP-seq barcodes (Bioo Scientific). Following elution of complexes from the Protein-A sepharose, crosslinks were reversed and barcoded libraries were amplified by PCR. The number of PCR cycles was determined empirically for each library. After amplification, library concentration was quantified using Qubit analysis and real time PCR.