title: ChIP-seq analysis of H-NS in Acinetobacter baumannii AB5075
design: ChIP-seq analysis of H-NS in Acinetobacter baumannii AB5075
library_construction_protocol: A. baumannii AB5075 is a clinical strain isolated from an infected tibia.Jacobs, A. C. et al. AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments. (2014) doi:10.1128/mBio.01076-14. Cells were grown in liquid LB medium at 37 degrees with shaking. Immunoprecipitations were done as described by Haycocks et al. (PLoS Genet. 2019. 15, e1008362) using A. baumannii strain AB5075. 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 (NEBNext Ultra II DNA Library Prep Kit for Illumina) and NEBNext Oligos for Illumina. 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 a Tapestation and real time PCR.