E-MTAB-6702:CflagwhiH_FLAGAb_IP_s
Illumina HiSeq 2000 sequencing; Identification by ChIP-Seq of the binding sites of the developmental protein WhiH in the genome of Streptomyces venezuelae
Identification by ChIP-Seq of the binding sites of the developmental protein WhiH in the genome of Streptomyces venezuelae
CflagwhiH_FLAGAb_IP_s
Using 'Redirect' PCR targeting (Gust et al., 2003; Gust et al., 2004), the whiH gene was replaced with a single apramycin resistance (apr) cassette in Streptomyces venezuelae (ATCC10712). An integrating plasmid, encoding a 3xFLAG-tagged version of WhiH, was then conjugated into the whiH mutant (SV8). The resulting strain was used in the subsequent ChIP-seq experiments, with the wild-type (ATCC10712) serving as a negative control. Both strains were grown as described in the growth protocol.  Cultures were grown in 4 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 (38 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 0.5 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.  Cell lysates were incubated with anti-FLAG M2 Affinity Gel (Sigma A2220) overnight at 4 degree C 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).  Library construction and sequencing were performed by The Genome Analysis Centre (TGAC), Norwich Research Park, Norwich, United Kingdom. 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 3 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 is 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.
Department of Molecular Microbiology John Innes Centre
E-MTAB-6702:CflagwhiH_FLAGAb_IP_s
Experimental Factor: genotype
FLAG-tagged WhiH expression
Experimental Factor: immunoprecipitate
anti-FLAG