title: Sigma_70_sampleA_1
organism: Escherichia coli ETEC H10407
description: Protocols: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2017-09-30T17:02:12Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2017-02-27T15:05:41Z
attributes.tag: External Id
attributes.value: SAMEA100988668
attributes.tag: INSDC center name
attributes.value: Institute of Microbiology and Infection School of Biosciences University of Birmingham
attributes.tag: INSDC first public
attributes.value: 2017-09-30T17:02:12Z
attributes.tag: INSDC last update
attributes.value: 2017-02-27T15:05:41Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5521:Sigma_70_sampleA_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: transformed with plasmid pRGM9818
attributes.tag: sample name
attributes.value: E-MTAB-5521:Sigma_70_sampleA_1
attributes.tag: scientific_name
attributes.value: Escherichia coli
attributes.tag: strain
attributes.value: H10407