GSM8381837: WT_LK; Caulobacter vibrioides; ChIP-Seq
Resuspended pellets were lysed with 2 µl of Ready-lyse lysozyme solution (Epicentre)  for 5 min at RT. Protease inhibitors (Roche) was added and incubated for 10 min. Then, 550 µl of ChIP buffer (1.1% triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, plus protease inhibitors) was added to the lysate and incubated at 37 °C for 10 min before sonication (2 x 8 bursts of 30 sec on ice using a Diagenode Bioruptor) to shear DNA fragments to a length of 300 to 500 bp. Lysate was cleared by centrifugation for 10 min at 12,500 rpm at 4 °C and protein content was evaluated by measuring OD280. Then, 7.5 mg of proteins were diluted in ChIP buffer supplemented with 0.01% SDS and precleared 1 h at 4 °C with 50 µl of protein A magneticbeads (BioRad) and 100 µg BSA. One µl of polyclonal anti-KdpE antibodies, produced by rabbits (CER Groupe, Marloie, Belgium) immunised with purified His6-KdpE protein, were added to the supernatant before overnight incubation at 4 °C under gentle agitation. Eighty µl of BSA pre-saturated protein A magnetic beads were added to the solution for 2 h at 4 °C with rotation, washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), once with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA) at 4 °C and a second wash with TE buffer at RT. The DNA-protein complexes were eluted twice in 250 µl freshly prepared elution buffer (0.1 M NaHCO3, 1% SDS). NaCl was added at a concentration of 300 mM to the combined eluates (500 µl) before overnight incubation at 65 °C to reverse the crosslink. The samples were treated with 20 µg of proteinase K in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5) for 2 h at 45 °C. DNA was extracted using Nucleospin PCR clean-up kit (Macherey-Nagel) and resuspended in 50 µl elution buffer (5 mM Tris-HCl pH 8.5). ChIP-Seq libraries were prepared at the Seqalis company (Belgium) using the Illumina TruSeq DNA library (Roche-KapaHyperPrep)  according to the manufacturer's instructions.