title: GSM2854968: wild type, dawn; Synechococcus elongatus PCC 7942; ChIP-Seq
library_construction_protocol: Samples were resuspended in 500 μl phosphate-buffered saline and pelleted for 5 min at 3,000 x g. The pellets were flash frozen and stored at -80 °C. Samples were thawed and resuspended in 270 μl of lysis buffer (50 mM HEPES-NaOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1x Roche Complete EDTA-free Protease Inhibitors). Cells were disrupted by 10 cycles of 30 sec bead-beating at 4 °C with 0.1 mm glass beads with periodic cooling on ice. Chromatin was sheared by sonication to a median length of ~300 bp. Lysates were then clarified by two rounds of centrifugation at 21,000 x g for 15 min at 4 °C. Protein concentration was determined by BCA assay (Pierce Biotechnology) with a standard curve prepared with Bovine Serum Albumin. 750 μg of lysate from each time point (prepared in 500 ul of the lysis buffer) were mixed with 18 μg of purified anti-E. coli RNA polymerase β monoclonal antibody (BioLegend). Samples were incubated overnight at 4 °C with continuous rotation. 70 μl of 1:1 mix of rProtein A Sepharose and Protein G Sepharose Fast Flow beads (GE Healthcare; 50% slurry in the lysis buffer) were added to each sample.  After 2 h of incubation, beads were isolated by centrifugation for 1 min at 1000 g, washed twice with lysis buffer, once with wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.1% sodium deoxycholate) and once with TE pH 7.5 (10 mM Tris-HCl pH 7.5, 1 mM EDTA). Elution of protein-DNA complexes was performed with 250 μl elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) for 1 h at 65 °C. Crosslinks were reversed by incubation of samples at 65 °C for 12 h. Proteins were digested with proteinase K for 2 h at 37 °C, and samples were extracted with phenol/chloroform/isoamyl alcohol. Nucleic acids were precipitated with ethanol overnight and RNA was digested in 50 μl of TE containing 20 ng/μl of DNase-free RNase (Fermentas). Samples were extracted again with phenol/chloroform/isoamyl alcohol, precipitated with ethanol and resuspended in 50 μl 10 mM Tris-HCl pH 8.0. Libraries for Illumina sequencing of ChIP DNA were prepared following a protocol developed by Ethan Ford (http://ethanomics.files.wordpress.com) with modifications as described in Markson et al., 2013.