| Construction Protocol | To obtain well-grown mycelia, spores (109) of S. coelicolor (WT and ∆hsr mutant) were inoculated in 10 ml R2 medium and grown as standing culture at 30°C for 16 h and afterwards on a rotary shaker for 16 h after the addition of 90 ml R2 medium. The cultures were washed four times in minimal medium without supplement. The mycelia were suspended in 50 ml R2 medium and divided in two 25 ml-portions, one of which contained H2O2 (0.15 mM). Cultivation was continued at 30 °C on a rotary shaker for two hours. Mycelia were harvested by centrifugation and the mycelia pellets were store at -80 °C. Total RNA was isolated from two biological replicates using in part a previously reported protocol Ortiz de Orue Lucana and Schrempf (2000) and the RNeasy Mini Kit along with a DNase Kit (both from Qiagen). Briefly, mycelia were washed with 25 mM Tris, pH 7.5. 1 g mycelia was resuspended in a 3 ml-solution of 4 M guanidinthiocyanate, 25 mM sodium citrate, pH 7, 0.5 % laurylsarcosine and 0.1 M 2-mercaptoethanol, and treated with 1 vol. glass beads by vortexing for 3 min. The glass beads and the cell debris were removed by centrifugation. 500 μl of the supernatant were used for isolation according to the protocols described in the RNeasy Mini and DNase Kit. The quality of the RNA was controlled on a 2 % agarose gel, and it was tested for possible DNA contaminations by PCR. RNA concentrations and quality were determined using Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Boblingen, Germany). Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to deplete ribosomal RNA. The quality of rRNA-depleted RNA samples was checked on Agilent 2100 Bioanalyzer. TruSeq® Stranded mRNA low sample protocolILLUMINA PROPRIETARYPart # 15031047 Rev. DSample Preparation Guide |
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