alias: E-MTAB-5521:MarA_sampleA_1_p
title: Illumina MiSeq paired end sequencing; ChIP-seq of the multiple antibiotic resistance activator (MarA) protein and RNA polymerase sigma70 subunit of Enterotoxigenic Escherichia coli strain H10407
design: ChIP-seq of the multiple antibiotic resistance activator (MarA) protein and RNA polymerase sigma70 subunit of Enterotoxigenic Escherichia coli strain H10407
library_construction_protocol: Prototypical enterotoxigenic Escherichia coli strain first isolated from a patient with cholera-like symptoms in Bangladesh prior to 1973 Cells were grown in liquid LB medium at 37 degrees with shaking Immunoprecipitations were done as described by Haycocks et al. (PLoS Pathog. 2015. 11:e1004605) using bacterial strain H10407 transformed with plasmid pRGM9818 encoding marA under the control of the tac promoter. This allowed us to circumvent repression of the chromosomal mar locus by MarR. After growing cells to midlog phase, crosslinking, and sonication, protein DNA complexes were immunoprecipitated using anti-sigma70, anti-MarA or anti-MarR. Anti-sigma70 was purchased from Neoclone (Madison, USA). Anti-MarA and anti-MarR were a kind gift from Lee Rosner and Bob Martin (NIH, Bethesda).   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.
identifiers.namespace: Institute of Microbiology and Infection School of Biosciences University of Birmingham
identifiers.id: E-MTAB-5521:MarA_sampleA_1_p
attributes.tag: Experimental Factor: Anti-MarA
attributes.value: immunoprecipitate