GSM2413408: popZ-KE_ZitP-N; Caulobacter vibrioides; ChIP-Seq
Mid-log phase cells were cross-linked in 10 mM sodium phosphate (pH 7.6) and 1%  formaldehyde at room temperature for 10 min and on ice for 30 min thereafter, washed  thrice in phosphate buffered saline (PBS) and lysed in a Ready-Lyse lysozyme solution  (Epicentre, Madison, WI) according to the manufacturer`s instructions. Lysates were  sonicated (Sonifier Cell Disruptor B-30; Branson Sonic Power. Co., Danbury, CT) on ice  using 10 bursts of 20 sec at output level 4.5 to shear DNA fragments to an average length  of 0.3-0.5 kbp and cleared by centrifugation at 14,000 rpm for 2 min at 4°C. Lysates were  normalized by protein content, diluted to 1 mL using ChIP buffer (0.01% SDS, 1.1%  Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl plus protease  inhibitors (Roche) and pre-cleared with 80 μL of protein-A agarose (Roche, Switzerland)  and 100 uμg BSA. Ten % of the supernatant was removed and used as total chromatin  input DNA as described before (Radhakrishnan et al., 2008).  Two microliters of polyclonal antibodies were added to the remains of the  supernatant, incubated overnight at 4°C with 80 μL of protein-A agarose beads pre  saturated with BSA, 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), high salt buffer (0.1% SDS, 1%  Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl) and LiCl buffer  (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH  8.1) and twice with TE buffer (10 mM Tris-HCl (pH 8.1) and 1 mM EDTA). The  protein•DNA complexes were eluted in 500 μL freshly prepared elution buffer (1% SDS,  0.1 M NaHCO3), supplemented with NaCl to a final concentration of 300 mM and  incubated overnight at 65°C to reverse the crosslinks. The samples were treated with 2 μg  of Proteinase K for 2 h at 45°C in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5). DNA  extracted using phenol:chloroform:453 isoamyl alcohol (25:24:1), ethanol-precipitated  using 20 ug glycogen as carrier and re-suspended in 100uL of water. For deep  sequencing (ChIP-seq) total chromatin input DNA was not saved. DNA libraries were prepared for sequencing using standard Illumina protocols by FASTERIS SA, Switzerland
GEO Accession
GSM2413408