Illumina HiSeq 2500 paired end sequencing; Brucella abortus ChipSeq analysis of BvrR
Brucella abortus ChipSeq analysis of BvrR
The wild-type and BvrR-deficient strains were cultured until the mid-logarithmic phase as described above. Before crosslinking, half of the pelleted bacteria were incubated in a nutrient-limited medium (33 mM KH2PO4, 60.3 mM K2HPO4, 0.1% yeast extract) at pH 5.5 adjusted with citric acid, for five minutes. The other half of the pelleted bacteria was incubated in fresh TSB for 5 minutes as control. Crosslinking was performed as indicated and stopped by adding glycine to a final concentration of 125 mM as described elsewhere. After adding lysis buffer with lysozyme at 10 mg/ml, bacteria were lysed in the cell Disruptor Genie from Scientific Industries at maximal amplitude (2800) for 1 h, at 4 °C, followed by overnight incubation with ChIP buffer at 37°C. The lysate was sonicated on ice (Branson Sonifier Digital cell disruptor S-450D 400W) by applying 25 bursts of 30 s at 30% amplitude and 30 s of pause. A polyclonal rabbit anti-BvrR antibody and magnetic beads coated with protein A were used for immunoprecipitation.  DNA was extracted using a standard protocol of isopropanol precipitation. Library construction (125 bp paired-end) and Illumina HiSeq 2500 HT sequencing were performed at Genomics Core Leuven, Belgium.