title: Illumina MiSeq sequencing; RNA-Seq of roseoflavin producer Streptomyces davaonensis reveals transcription of unusual high number of riboflavin biosynthetic genes (rib genes) in exponential and stationary growth phase
design: RNA-Seq of roseoflavin producer Streptomyces davaonensis reveals transcription of unusual high number of riboflavin biosynthetic genes (rib genes) in exponential and stationary growth phase
library_construction_protocol: S. davaonensis was grown in YS and cells were harvested in the exponential growth phase (after 18 h of growth) and from the stationary growth phase (after 48 h of growth). For each condition three independent cultures inoculated with the same amount of spores were analyzed.  S. davaonensis was grown in YS and cells were harvested in the exponential growth phase (after 18 h of growth) and from the stationary growth phase (after 48 h of growth). For each condition three independent cultures inoculated with the same amount of spores were analyzed.  Total RNA was isolated from three biological replicates using RNeasy Mini Kit along with a DNase Kit (both from Qiagen). For primary 5′-end-enriched cDNA libraries, rRNA-depleted RNA samples obtained from 2 × 5μg of total RNA were used. The cDNA library preparation was performed as originally described in Peifer-Sancar et al. (2013) with the additional modifications to strongly reduce the number of false positives described in Kranz et al. 2018. In brief, rRNA-depleted mRNA was treated with 5′-monophosphate-dependent exonuclease to digest transcripts with 5'-monophosphates. Undigested transcripts were ligated to an RNA 5′-index adapter. Conversion of true primary 5′-triphosphate ends to 5′-monophosphate ends with RNA 5′-polyphosphatase (RPP) treatment prepared the transcripts for 5′-adapter ligation. Reverse transcription with a stem-loop DNA adapter and library amplification was performed as described  Kranz et al. 2018