Illumina HiSeq 2500 sequencing; Identification by ChIP-Seq of the binding sites of the sigma factor WhiG in the genome of Streptomyces venezuelae
Identification by ChIP-Seq of the binding sites of the sigma factor WhiG in the genome of Streptomyces venezuelae
Streptomyces venezuelae strain NRRL B-65442 (NCBI Ref: NZ_CP018074.1) was used as the wild type strain. The WhiG deletion strain (SV6) was made using 'Redirect' PCR targeting (Gust et al., 2003; Gust et al., 2004), to replace the whiG gene with a single apramycin resistance (apr) cassette in the wild type Streptomyces venezuelae strain.  Cultures were grown in 2 x 30 ml volumes of MYM-TAP according to Stuttard C. 1982. (Microbiology 128:115121) made with 50% tap water and supplemented with 200 ul of trace element solution (Kieser T et al. 2000. Practical Streptomyces genetics. John Innes Foundation, Norwich, United Kingdom.) per 100 ml. Cultures were incubated at 30 degrees c and 250 RPM until sporulation was at an advanced stage (34 hours, as determined by light microscopy).  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 0.75 ml of lysis buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl), containing 15 mg/ml of lysozyme and 1x protease inhibitor (Roche Applied Science) and incubated at 37 degree 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. The samples were sonicated for 8 cycles of 20 s each at 8 micrometer to shear chromosomal DNA into fragments ranging from 300 to 1,000 bp on average. The samples were centrifuged twice at 16,000g at 4 degree C for 10 min to clear the cell extracts.  Cell lysates were incubated with 10% (v/v) protein A-sepharose (Sigma) for 1 h on a rotating wheel to remove non-specifically binding proteins. Samples were then centrifuged for 15 min at 4ºC and 13,000 rpm to remove the beads. Supernatants were incubated with 10% (v/v) anti-WhiG antibody (Polyclonal from rabbit; Cambridge Research Biochemicals) overnight at 4 ºC with rotation. Subsequently, 10% (v/v) protein A-sepharose was added to precipitate WhiG and incubation was continued for 4 h. Samples were centrifuged at 3500 rpm for 5 min and the pellets were washed twice with 0.5x IP buffer, and then twice with 1x IP buffer (100 mM Tris HCl pH 8, 250 mM NaCl, 0.5% Triton-X-100, 0.1% SDS). Each pellet was incubated overnight at 65 ºC in 150 µl IP elution buffer (50 mM Tris HCl pH 7.6, 10 mM EDTA, 1% SDS) to reverse cross-links. Samples were centrifuged at 13,000 rpm for 5 min to remove the beads. Each pellet was re-extracted with 50 µl TE buffer (10 mM Tris HCl pH 7.4, 1 mM EDTA) and the supernatant incubated with 0.2 mg/ml Proteinase K (Roche) for 2 h at 55ºC. The resulting samples were extracted with phenol-chloroform and further purified using QiaQuick columns, eluting in 50 µl EB buffer (Qiagen).  For each sample, 10ng of DNA was used for library preparation using NEBNext ChIP-Seq Library Prep Kit following their recommended procedure.
Experimental Factor: immunoprecipitate
none
Experimental Factor: genotype
whiG knockout