title: AphA C-terminal 3xFLAG sample A
organism: Vibrio cholerae
description: Protocols: The Vibrio cholerae O1 El Tor strain E7946 was isolated in Brazil (Levine MM, Black RE, Clements ML, Cisneros L, Saah A, Nalin DR, et al. The pathogenicity of nonenterotoxigenic Vibrio cholerae serogroup O1 biotype El Tor isolated from sewage water in Brazil. J Infect Dis. 1982;145: 296-299). 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. 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: 2019-10-21T17:04:14Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-05-10T11:13:33Z
attributes.tag: External Id
attributes.value: SAMEA5602943
attributes.tag: INSDC center name
attributes.value: School of Biosciences, University of Birmingham
attributes.tag: INSDC first public
attributes.value: 2019-10-21T17:04:14Z
attributes.tag: INSDC last update
attributes.value: 2019-05-10T11:13:33Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7953:AphA C-terminal 3xFLAG sample A
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: developmental stage
attributes.value: mid logarithmic growth
attributes.tag: genotype
attributes.value: wild type
attributes.tag: organism part
attributes.value: nucleoid
attributes.tag: plasmid
attributes.value: pAMCFaphA
attributes.tag: sample name
attributes.value: E-MTAB-7953:AphA C-terminal 3xFLAG sample A
attributes.tag: scientific_name
attributes.value: Vibrio cholerae
attributes.tag: strain
attributes.value: E7946