Chip-seq analysis of NusA and Rho binding in Escherichia coli with or without H-NS
Chip-seq analysis of NusA and Rho binding in Escherichia coli with or without H-NS
Bacterial strain MG1655 is a laboratory strain of Escherichia coli.  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 MG1655. After growing cells to midlog phase, crosslinking, and sonication, protein DNA complexes were immunoprecipitated using anti-NusA or anti-Rho. 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.