GSM4656002: Chip 3; Pseudomonas aeruginosa; ChIP-Seq
DNA was sonicated to shear DNA to an average size of 200–500 bp.Insoluble cellular debris was removed by centrifugation and the supernatant was saved. Immunoprecipitation samples were incubated overnight at 4°C on a rotating wheel with protein A beads (Cat#: SA015), which was incubated with 100 μl agarose-conjugated anti-flag antibodies (Cat#: AGM12165, Aogma) in IP buffer. Input samples without antibody were set up as negative control. Washing, crosslink reversal, and purification of the ChIP DNA were conducted by following previously published protocols with minor changes. Briefly, the beads were then collected and washed once with 1 ml of IP buffer, once with 1 ml of IP buffer plus 500 mM NaCl, once with 1 ml of IP buffer plus 250 mM LiCl, and once with 1 ml of Tris-EDTA buffer (pH 7.5). Immunoprecipitated complexes were eluted from the beads by treatment with 100 μl elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) at 65°C for 20 min. Samples were then treated with Protease K (TIANGEN) and cross-links were reversed by incubation overnight at 65°C. Overnight samples were then treated with 50 μg RNase A at 37°C for 1 h. DNA was extracted twice with phenol-chloroform, precipitated with ethanol at -80°C for 4 h, and then resuspended in 20 μl of deionized water. DNA fragments (200 to 500 bp) were selected for library construction, and sequencing libraries prepared using the NEBNext Ultra II DNA Library Prep kit. The final DNA libraries were validated with Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit, then the libraries were sequenced using the HiSeq PE150 system (Illumina).