alias: E-MTAB-5591:MarR-8xmyc_sample1_s
title: Illumina MiSeq sequencing; ChIP-seq of the multiple antibiotic resistance repressor (MarR) protein of Enterotoxigenic Escherichia coli strain H10407
design: ChIP-seq of the multiple antibiotic resistance repressor (MarR) protein 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 plasmids pAMNF or pAMNM encoding MarR N-terminally fused to 3xFLAG or 8xmyc respectively. After first growing cells to midlog phase, crosslinking, and sonication, protein DNA complexes were immunoprecipitated using anti-FLAG (Sigma). 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-5591:MarR-8xmyc_sample1_s
attributes.tag: Experimental Factor: expression of MarR-8xmyc fusion protein
attributes.value: phenotype