PA1290 mutant complement TY1
The ChIP-seq assays were performed as previously described (Heacock-Kang, 2018). Briefly, the regulator mutant strain PA1290Tn containing IPlac-3xTY1-PA1290 was grown and induced by isopropyl -D-1-thiogalactopyranoside (IPTG) (0.25 mM) in LB at 30 C for 48h without shaking. After induction, bacterial cells were cross-linked with paraformaldehyde, harvested, and lysed by freezing and thawing. Supernatants containing the DNA-regulator complexes were sheared to an average size of 100500 bp in a Roche nebulizer. The TY1-tagged PA1290 regulator bound to its regulated DNA region/fragment was enriched and captured using TY1 mouse monoclonal antibody and DiaMag anti-mouse IgG-coated magnetic beads (Diagenode), respectively. Captured DNA-regulator complexes were reversed cross-linked and purified using the QIAquick PCR Purification Kit (QIAGEN). Purified DNA fragments were used as a template for library construction according to the Illumina ChIP-seq sample preparation protocol and sequenced at TUCF Genomics, where DNA libraries were prepped and 50 bp single-end reads were sequenced with the Illumina HiSeq 2500.