title: ChIP-seq experiment of ParB binding to Streptomyces venezuelae chromosome in wild type and SMC knockout strain
design: ChIP-seq experiment of ParB binding to Streptomyces venezuelae chromosome in wild type and SMC knockout strain
library_construction_protocol: 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