GSM6523948: pRAB11; Staphylococcus aureus; ChIP-Seq
Bacterial cells were suspended in 1ml of lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, 100 μg / ml lysostaphin (Ambi), 500 µg / ml RNaseA, complete mini protease inhibitor EDTA free containing 100 um zirconium beads) and incubated for 20 min at 37°C. The weakened cells were then disrupted using bead beating at 6000 rpm for 1 min and the cell lysate clarified by centrifugation (20,000xg/10min/4°C). The cation concentration of the lysate was adjusted with MgCl2 and CaCl2 to 100 mM. The DNA was sheared by the addition of a range of DNase I (NEB) concentrations (2 – 0.5 U), in a 200 uL volume, followed by incubation at 37°C for 10 min. The reaction was quenched by the addition of EDTA to 50 mM on ice. The degree of DNA fragmentation was assessed by electrophoresis of samples on a 2% (v/v) agarose TAE gel. Samples showing maximal fragmentation at between 100-300 bp were subjected to immunoprecipitation. Lysis buffer (made up to 4 ml - omitting lysostaphin and RNaseA) containing 10 µg of M2-anti FLAG antibody was added and incubated overnight at 4°C on a rotating platform in a 15 ml tube. A 100 ul of Protein G agarose (Pierce) was added to the lysate and incubated a further 2h at room temperature. The sample was centrifuged (2,500 xg/3 min/22°C) and the agarose pellet washed three time with 4 ml of IP buffer (25mM Tris.Cl [pH 7.2}, 150mM NaCl) and finally resuspended in 200 ul of elution buffer (10mM Tris.Cl [pH 8], 1mM EDTA, 1% SDS containing 100µg of proteinase K). Crosslinks were reversed incubation for 2 h at 37°C and then 9 h at 65°C with shaking at 1400 rpm. Finally, eluted DNA was cleaned up by PCR purification (QiaQuick PCR purification kit, Qiagen). DNA libraries were prepared with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina and sequenced using an Illumina MiSeq.