ChIP-seq experiment of ParB binding to Streptomyces venezuelae chromosome in wild type and SMC knockout strain
ChIP-seq experiment of ParB binding to Streptomyces venezuelae chromosome in wild type and SMC knockout strain
S. venezuelae strains were cultures in 50 ml liquid MYM medium supplemented with Trace Element Solution (TES, 0.2x culture volume)53 at 30°C with shaking. Cultures were inoculated with 100 ml of spores suspension with OD600=10. After 5 h of growth, the cultures were cross-linked with 1% formaldehyde for 30 min followed by blocking for 5 min with 125 mM glycine. Next, half of the culture volume was washed twice with PBS buffer, and the pellet was resuspended in 750 l of lysis buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 14 mg/ml lysozyme, protease inhibitor (Pierce)). After 1 h incubation at 37°C for 14 h, 200 μl of zircon beads (0.1 mm, BioSpec products) were added and the samples were disrupted using a FastPrep-24 Classic Instrument (MP Biomedicals; 2 x 45 s cycles at 6 m/s speed with 5-min breaks, during which the lysates were incubated on ice). Next, cell lysates were mixed with 750 l IP buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.8% Triton X-100, a protease inhibitor (Pierce)), and sonicated to shear DNA into fragments ranging from 300 to 500 bp. 50 ml liquid MYM medium supplemented with TES at 30°C, 200 rpm Immunoprecipitation was performed overnight at 4°C. Next, the magnetic beads were washed twice with IP buffer, once with IP2 buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.8% Triton X-100, protease inhibitors (Pierce)), and once with TE buffer (Tris-HCl, pH 7.6, 10 mM EDTA 10 mM). After washing, magnetic beads were resuspended in IP elution buffer (50 mM Tris-HCl pH 7.6, 10 mM EDTA, 1% SDS) an incubated overnight at 65°C. Next, the samples were centrifuged, and proteinase K (Roche) was added to the supernatants to a final concentration of 100 µg/ml followed by incubation for 90 min at 55°C. DNA was extracted with phenol and chloroform and subsequently precipitated overnight with ethanol. The precipitated DNA was dissolved in nuclease-free water (10 µl).  Libraries were prepared by Fasteris SA (Switzerland) according to ChIP-seq protocol