title: Illumina HiSeq 2000 sequencing; Genes required for aerial growth, cell division, and chromosome segregation are targets of WhiA before sporulation in Streptomyces venezuelae.
design: Genes required for aerial growth, cell division, and chromosome segregation are targets of WhiA before sporulation in Streptomyces venezuelae.
library_name: Flag40hTot
library_construction_protocol: S. venezuelae strains were cultured in MYM-TAP according to Stuttard C. 1982. (Microbiology 128:115–121) made with 50% tap water and supplemented with 200 μl of trace element solution (Kieser T et al. 2000. Practical Streptomyces genetics. John Innes Foundation, Norwich, United Kingdom.) per 100 ml. Conjugations between E. coli and S.  venezuelae were essentially carried out as described for S. coelicolor (Kieser T et al. 2000. Practical Streptomyces genetics. John Innes Foundation, Norwich, United Kingdom).  However, because S. venezuelae grows much more quickly than S. coelicolor, the plates were incubated either at room temperature overnight or at 30°C for only 7 h before overlaying with the selective antibiotics. 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.
attributes.tag: Experimental Factor: input DNA
attributes.value: immunoprecipitate
attributes.tag: Experimental Factor: FLAG-WhiA
attributes.value: genotype