Illumina MiSeq paired end sequencing; ChIP-seq of the cyclic-AMP Receptor Protein (CRP) and RNA polymerase sigma 70 subunit in Vibrio cholerae
ChIP-seq of the cyclic-AMP Receptor Protein (CRP) and RNA polymerase sigma 70 subunit in Vibrio cholerae
The Vibrio cholerae O1 El Tor strain N16961 was isolated in 1971 in Bangladesh. Cells were grown in liquid M9 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 N16961. After growing cells to midlog phase, crosslinking, and sonication, protein DNA complexes were immunoprecipitated using anti-sigma70, anti-CRP or no antibody as a control. Anti-sigma70 was purchased from Neoclone (Madison, USA). Anti-CRP was as described previously (Greinger et al. 2005. Proc. Natl. Acad. Sci. USA. 102:17693-8.).   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.
Experimental Factor: immunoprecipitate
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