Illumina HiSeq 2000 sequencing; ChIP-Seq to determine binding sites of transcription factor BldO in the genome of Streptomyces venezuelae
ChIP-Seq to determine binding sites of transcription factor BldO in the genome of Streptomyces venezuelae
Using 'Redirect' PCR targeting (Gust et al., 2003; Gust et al., 2004), the bldO gene was replaced with a single apramycin resistance (apr) cassette in Streptomyces venezuelae (ATCC10712). An integrating plasmid, encoding a 3xFLAG-tagged version of BldO, was then conjugated into the bldO mutant. The resulting strain was used in the subsequent ChIP-seq experiments, with the wild-type (ATCC10712) serving as a negative control. 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.