title: Comparative evaluation of H-NS binding profiles of three E. coli strains
design: Comparative evaluation of H-NS binding profiles of three E. coli strains
library_name: E. coli K-12 (W3110)-H-NS-His12_WCE_First
library_construction_protocol: ChAP (chromatin affinity precipitation) was performed basically as original procedure (Ishikawa et al., 2007). Briefly, all E. coli strains grow in 50 ml of LB media under aerobic condition at 37 ?C. Protein-DNA complex are fixed by adding formaldehyde (final concentration is 1.0 %.) to the bacterial culture at OD600 ? 0.4 and incubating at room temperature for 20 minutes. Then, quenching reaction was performed by the addition of 3M Glycine solution (final concentration is 90 mM) followed by incubation at room temperature for 5 minutes. After fixation, cells were harvested by centrifugation, and washed by TBS buffer and stored at -80 ?C until use. Cells were disrupted by sonication on ice in 3 mL of UT buffer (100 mM HEPES, 50 mM imidazole, 8 M urea, 0.5 M NaCl, 1% Triton X-100, 10mM ?-mercaptoethanol, 1 mM PMSF, pH 7.4). After sonication, samples are centrifuged at 14000 rpm for 10 minutes to remove cell debris. Aliquot (100 ? 200 ?l) of supernatant were took for the fraction of WCE (whole cell extract) and remaining extract were mixed with 50 ?l of Dynabeads Talon (Invitrogen, Norway). Mixtures were rotated at 4 ?C for overnight. Talon was washed five times with UT buffer, and bound protein-DNA complexes were eluted with 400 ?L of elution buffer (100 mM Tris?HCl, pH 7.5, 0.5 M imidazole, 1% SDS, 10 mM DTT). The eluate was passed through Microcon-100 (Millipore, Ireland) to remove nonspecifically bound uncross-linked proteins with a molecular mass lower than 100 kDa. DNA-Protein complexes retained on the membrane were washed three times with wash buffer (100 mM Tris?HCl, pH 7.5, 1% SDS, 10 mM DTT), and recovered by the addition of 50 ?L buffer  (ChAP fraction). Cross-links between DNA and protein in ChAP and WCE fraction were dissociated by heating at 65 ?C overnight, and DNA was purified using Qiaquick (QIAGEN, Germany). Using the recovered DNA, the samples were prepared for the Illumina GA according to the manufacturer's instructions.