Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of Streptomyces venezuelae grown overnight (14 hrs) with Mitomycin-C against untreated controls.
RNA-Seq of Streptomyces venezuelae grown overnight (14 hrs) with Mitomycin-C against untreated controls.
Mycelial mass was harvested by centrifugation for 30 ml cultures grown overnight (14 hours). Streptomyces venezuelae NRRL B-65442 was grown in triplicates for 17 hours in 30 ml shaken cultures at 30 degrees Celsius in the Maltose-Yeast extract-Malt Extract (MYM) medium supplemented with R2 trace element solution containing 0.25 ug/ml Mitomycin C (MMC). Controls were also grown similarly in triplicates except that the medium did not contain any MMC. Mitomycin C was added to the growth medium at 2.5 ug per ml. The .bam files produced by aligning the reads to the genome were used as input to the featureCounts tool of the Bioconductor package Rsubread (version 2.0.0) along with a a .saf file containing coordinates of genes in the genome. This produced a table of counts of reads mapping to every gene in the genome. Mycelial pellets were washed with 1x PBS followed by lysis and RNA purification using the RNEasy kit (Quiagen). Purified RNA samples were treated with on-column DNase I (Qiagen), followed by an additional DNase I treatment (Turbo DNA-free, Invitrogen). rRNA depletion and Library construction were carried out by Genewiz (NJ, USA).