Illumina HiSeq 2000 paired end sequencing; ChIP-Seq with anti-FLAG antibodies to determine binding of LexA-3xFLAG to DNA in Streptomyces venezuelae treated with Mitomycin C (MMC) overnight
ChIP-Seq with anti-FLAG antibodies to determine binding of LexA-3xFLAG to DNA in Streptomyces venezuelae treated with Mitomycin C (MMC) overnight
Formaldehyde was added to cultures at a final concentration of 1% (vol/vol), and incubation was continued for 30 min. Glycine was then added to a final concentration of 125 mM to stop the cross-linking. The samples were left at room temperature for 5 min and washed twice in phosphate-buffered saline (PBS) buffer (pH 7.4). The pellets were resuspended in 0.5 ml of lysis buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl), containing 15 mg ml−1 of lysozyme and 1x protease inhibitor (Roche Applied Science) and incubated at 37°C for 25 min or until lysed. Subsequently, 0.5 ml immunoprecipitation (IP) buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 0.8% [vol/vol] Triton X-100), containing 1x protease inhibitor, was added, and the samples were chilled on ice.  S. venezuelae NRRL B-65442 was grown in maltose-yeast extract-malt extract medium (MYM), composed of 50% tap water and 50% reverse osmosis water, and supplemented with R2 trace element (TE) solution at a ratio of 1:500. Liquid cultures were grown under aeration at 30°C and at 250 rpm. MYM agar was supplemented with the following antibiotics when required: 5 μg/ml kanamycin, 25 μg/ml hygromycin, or 50 μg/ml apramycin. Cells were grown in MYM medium in the absence (untreated) and presence of 0.25 µg/ml MMC for 14h (14-MMC) at 30°C. The samples were sonicated for six cycles of 20 s each at 10 µm to shear chromosomal DNA into fragments ranging from 300 to 1,000 bp on average. The samples were centrifuged twice at 16,000 x g at 4°C for 10 min to clear the cell extracts, after which 50 µl of each was set aside for total-DNA extraction. The remaining lysates were added to M2 (Sigma-Aldrich A2220) gel suspension (40 µl per ml of lysate), prepared and washed as described in the manufacturer's instructions. The mixtures were then incubated on a rotating wheel at 4°C overnight. The samples were centrifuged for 30 s at 4°C and 5,500 × g, and the pellets were washed twice with 0.5× IP buffer and then twice with 1× IP buffer and transferred to new Eppendorf tubes after the first washing step. The pellets and 50 µl of total cell extracts (set aside earlier) were eluted overnight at 65°C in 100 µl of IP elution buffer (50 mM Tris-Cl, pH 7.6, 10 mM EDTA, 1% SDS) to reverse the cross-links. The samples were centrifuged at 16,000 × g for 5 min to remove the beads. The pellets were reextracted with 50 µl of TE buffer (10 mM Tris-Cl, pH 7.4, 1 mM EDTA) and incubated with 0.2 mg ml−1 proteinase K (Roche) for 2 h at 55°C. The samples were extracted twice with phenol-chloroform and once with chloroform and further purified using QiaQuick columns (Qiagen). DNA was eluted in 50 µl EB buffer (10 mM Tris-Cl, pH 8.5) and quantified using a NanoDrop spectrophotometer (Thermo Scientific).  Library preparation was carried out by an external contractor (Genewiz UK Ltd.) using the illumina TruSeq ChIP Library Preparation Kit following manufacturers instruction.