GSM8478994: P. putida, hexR_8xMYC tag, M9 acetate, bioreplicate 1; Pseudomonas putida; OTHER
Cells at the mid–log phase were harvested and cross–linked with 1% formaldehyde at room temperature. Cells were lysed using lysozyme, and genomic DNA was sheared by sonication. Immunoprecipitation was performed using antibodies that specifically recognize the myc tag (9E10, Santa Cruz Biotechnology), and Dynabeads Pan Mouse IgG magnetic beads (Invitrogen). Briefly, IP DNA was repaired by the NEBNext End Repair Module (New England Biolabs). This is followed by an addition of a single dA overhang using dA–Tailing Module (New England Biolabs), ligation of the first adaptor (5′–phosphorylated) using NEBNext Quick Ligation Module (New England Biolabs), and treatment with PreCR Repair Mix (New England Biolabs). Lambda exonuclease– and RecJf exonuclease–treated chromatin was eluted from the beads and the protein–DNA crosslink was reversed by an overnight incubation at 65°C. Sequencing library construction was performed using purified DNA samples. This process includes the second strand synthesis, dA–tailing, second adapter ligation, 3' overhang removal, and PCR enrichment. The amplified DNA samples were purified again using the GeneRead Size Selection Kit (Qiagen) and quantified using Qubit dsDNA HS Assay Kit (Life Technologies). Library size distribution and quality were confirmed using Agilent High Sensitivity DNA Kit on Agilent 2100 Bioanalyzer (Agilent) before sequencing with NextSeq 500/550 or HiSeq X (Illumina). ChIP–exo experiments were conducted in biological replicates.