title: Illumina MiSeq sequencing; ChIP-seq analysis of Escherichia coli sigma 38 binding in cells lacking H-NS
design: ChIP-seq analysis of Escherichia coli sigma 38 binding in cells lacking H-NS
library_construction_protocol: Bacterial strain RPB104 is a laboratory strain of Escherichia coli 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 RPB104 delta hns. After first growing cells to stationary 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.