Illumina HiSeq 2000 sequencing; SvenBldMWhiIChIPSeq
SvenBldMWhiIChIPSeq
S. venezuelae strains were grown at 30 degree Celsius in MYM liquid sporulation medium (Bibb et al. Mol Microbiol 84: 1033-1049) made with 50% tap water and supplemented with 200 ul trace element solution (Kieser et al. Practical Streptomyces Genetics. The John Innes Foundation. Norwich, United Kingdom) per 100 ml. The phenotypes of mutants and complemented strains were scored after 3-4 days growth on MYM-agar at 30 degree Celsius. bldM and whiI deletion mutants were constructed by Redirect PCR targeting (Gust et al. Proc Natl Acad Sci U S A 100: 1541-1546) and their chromosomal structures were confirmed by PCR analysis and by Southern hybridization using the parental cosmids as probes. 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 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 six cycles of 20 s each at 10 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. For total DNA extraction, 50 microlitres of the cell extracts were incubated with 0.2 mg/ml proteinase K (Roche) for 2 h at 55 degree C, then extracted twice with phenol-chloroform and once with chloroform and further purified using QiaQuick columns (Qiagen). DNA was eluted in 50 microlitres EB buffer (10 mM Tris-Cl, pH 8.5) and quantified using a NanoDrop spectrophotometer (Thermo Scientific). Cell lysates were incubated with an anti-BldM or anti-WhiI polyclonal antibody and the antigen-antibody complexes were precipitated by the addition of protein-A Sepharose beads followed by centrifugation for 30 s at 4 degree C and 5,500g.  The pellets were washed twice with 0.5x IP buffer and then twice with 1x IP buffer and transferred to new Eppendorf tubes after the first washing step. The pellets and 50 microlitres of total cell extracts (set aside earlier) were eluted overnight at 65 degree C in 100 microlitres 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 16,000g for 5 min to remove the beads. The pellets were reextracted with 50 microlitres of TE buffer (10 mM Tris-Cl, pH 7.4, 1 mM EDTA) and incubated with 0.2 mg/ml proteinase K (Roche) for 2 h at 55 degree 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 microlitres EB buffer (10 mM Tris-Cl, pH 8.5) and quantified using a NanoDrop spectrophotometer (Thermo Scientific).  The TruSeq ChIP sample preparation kit from Illumina Inc. (part 15023092, revision A, August 2012) was used according to the recommended protocol. In brief, 5 to 10 ng ChIP DNA is blunt ended and phosphorylated, and a single A nucleotide is added to the 3prime ends of the fragments in preparation for ligation to an adapter with a single-base T overhang. The ligation products are purified and accurately size selected by agarose gel electrophoresis. Size-selected DNA was then purified and PCR amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantified using a combination of Bioanalyzer DNA HS Chip from Agilent Technology 2100 Bioanalyzer and Qubit 2.0 from Invitrogen.  The libraries were normalized to 10 nM and pooled, and quantitative PCR (q-PCR) was performed prior to cluster generation.  Each library pool was diluted to 2 nM with NaOH, and 5 microlitres was transferred into 995 microlitres HT-1 to give a final concentration of 10 pM.
Experimental Factor: genotype
bldM deletion
Experimental Factor: immunoprecipitate
anti-bldM antibody