Illumina HiSeq 3000 paired end sequencing; ChIP-seq experiment of HupS-FLAG binding to Streptomyces venezuelae chromosome in wild type and SMC knockout strain
After 14 hours of growth, the cultures were crosslinked with 1% formaldehyde for 30 minutes and blocked with 125 mM glycine. The cultures were washed twice with PBS buffer and the pellet obtained from half of the culture volume 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)) and incubated at 37°C for one hour. Next, 200 μl of zircon beads were added to the samples, which were further disrupted using FastPrep-24 Classic Instrument (MP Biomedicals;2 x 45 sec cycles at 6 m/s speed with 5-minutes breaks during which the lysates were incubated on ice). Next, the 750 l IP buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.8% Triton X-100, protease inhibitor (Pierce)) was added and the samples were sonicated to shear DNA into fragments ranging from 300 to 500 bp (controlled by agarose gel electrophoresis). The samples were centrifuged, 25 l was stored to be used as control samples (“input”) and the remaining supernatant was used for chromatin immunoprecipitation and mixed with the magnetic beads (60 l) coated with Anti-FLAG M2 antibody (Merck). Immunoprecipitation was carried out 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). 50 ml liquid MYM medium supplemented with TES at 30°C, 200 rpm DNA was released by overnight incubation of beads resuspended in the IP elution buffer (50 mM Tris-HCl pH 7.6, 10 mM EDTA, 1% SDS) at 65°C. The IP elution buffer was also added to the „input” samples which were treated further as immunoprecipitated samples. Next, the samples were centrifuged, and the proteinase K (Roche) was added to the supernatants to the final concentration of 100 µg/ml, followed by incubation for 90 min at 55°C. DNA was extracted with phenol and with chloroform and then precipitated overnight with ethanol. The precipitated DNA was dissolved  in nuclease-free water (10 µl). Libraries were prepared by Fasteris SA (Switzerland) acconrding ChIP-seq protocol