ChIP-seq analysis of MarA, SoxS, Rob and RamA biding across the Salmonella Typhimurium SL1344 genome
ChIP-seq analysis of MarA, SoxS, Rob and RamA biding across the Salmonella Typhimurium SL1344 genome
Salmonella enterica subsp. enterica serovar Typhimurium SL1344 is a histidine auxotroph of parental strain ST4/74. Hoiseth,S.K. and Stocker,B.A.D. (1981) Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature, 291, 238-239.  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 or anti-Myc.   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.