Illumina HiSeq 4000 paired end sequencing; Differential gene expression analysis by RNA-Seq for the bldC deletion strain and the wild type of Streptomyces venezuelae strain NRRL B-65442 after 10 and 14 hours of culture
Differential gene expression analysis by RNA-Seq for the bldC deletion strain and the wild type of Streptomyces venezuelae strain NRRL B-65442 after 10 and 14 hours of culture
Streptomyces venezuelae strain NRRL B-65442 (NCBI Ref: NZ_CP018074.1) was used as the wild type strain. The BldC deletion strain (SV25) was made using 'Redirect' PCR targeting (Gust et al., 2003; Gust et al., 2004), to replace the bldC gene with a single apramycin resistance (apr) cassette in the wild type Streptomyces venezuelae strain. 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). Mycelial pellets from duplicate MYM cultures were washed in PBS and resuspended in 900 μl lysis solution (400 μl phenol [pH4.3], 100 μl chlorophorm:isoamyl alcohol (24:1) and 400 μl RLT buffer [Qiagen]) with lysing matrix B (MP Biomedicals) and homogenised using a FastPrep FP120 Cell Disruptor (Thermo Savant). Two pulses of 30 seconds of intensity 6.0 were applied with cooling down for 1 min on ice between pulses. Supernatants were centrifuged for 15 min, full-speed on a bench-top centrifuge at 4°C and then treated according to the instructions given in the RNEasy Kit (Qiagen). The RNA samples were treated with on-column DNase I (Qiagen), followed by an additional DNase I treatment (Turbo DNA-free, Ambion) until they were free of DNA contamination (determined by PCR amplification of hrdB). Library preparation was performed by the sequencing contractor Genewiz (NJ, USA).
Experimental Factor: genotype
bldC knockout
Experimental Factor: time
14