title: RNA-seq of coding RNA of Actinoplanes sp. SE50/110 to analyze gene function of alternative sigma factor gene ACSP50_0507.
design: RNA-seq of coding RNA of Actinoplanes sp. SE50/110 to analyze gene function of alternative sigma factor gene ACSP50_0507.
library_construction_protocol: During cultivation, samples of 2x1 mL were taken and subsequently frozen in liquid nitrogen and stored at -80 °C until further RNA isolation  For the pre-culture, 30 mL NBS were inoculated from 200 µL glycerol stock. After cultivation for 72 h at 28°C and 140 rpm, 0.5 mL culture was passaged to a 30 mL new media and cultivated for another 48 h. Shake flask cultivation was performed using 250 mL Corning® Erlenmeyer baffled cell culture flasks for 50 mL culture at 28 °C and 140 rpm in maltose minimal medium ( (1) 72.06 g L−1 maltose·1H2O, (2) 5 g L−1 (NH4)2SO4, 0.184 g L−1 FeCl3·4H2O, 5.7 g L−1 Na3C6H5O7·2H2O, 1 g L−1, 200 µL trace elements (stock concentration: 1 μM CuCl2, 50 μM ZnCl2, 7.5 μM MnCl2), (3) MgCl2·6H2O, 2 g L−1 CaCl2·2H2O, and (4) 5 g L−1 each K2HPO4 and KH2PO4). For its preparation four medium components were separately prepared and autoclaved, whereas component (1) was sterile filtrated.  The inoculation was performed using, 30 mL pre-culture which was harvested by centrifugation at 5,000 rpm for 5 min and washed twice with maltose minimal medium. The cell wet weight was measured, and the cells were diluted to 50 g mL-1 of which 0.5 mL were used to inoculate 50 mL maltose minimal medium. The cultivation was performed for 7-9 days. Growth was determined by cell dry weight of 2x1 mL culture in pre-weight 1.5 mL reaction tubes, centrifugation at 14,000 rpm for 5 min and washed with H2O (MilliQ) twice before a 48 h drying process at 60°C. Zymo, RNA Clean & Concentrator R1014 and Quick-DNA Fungal/Bacterial Miniprep Kit D6005 (Zymo Research Corporation, Irvine, CA, USA) according to the user protocol. Cell disruption was performed of 1 mL cultivation sample at 2x 2600 for 30 sec with 5 min on ice in between. Disrupted samples were then centrifuged for 4 min at 4,000 rpm at 4°C. The DNaseI digest was performed with 75 µL Digestion Buffer and 5 µL enzyme for 15 min at room temperature and RNA was eluted with 87 µL RNase free H2O (MilliQ).   Control PCR to amplify remaining DNA was performed with genomic context binding oligonucleotides and Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 62°C for 30 sec and following agarose gel electrophoresis. If required, an additional DNaseI treatment of 15 min at RT was performed with 85 µL samples, 10 µL Digestion Buffer and 5 µL DNaseI. RNA elution was done afterwards with 30 µL RNase free H2O. RNA concentration and purity was analyzed using NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany).  5'-Seq cDNA library enriched for primary 5′-transcripts according to Peifer-Sancar et al. (2013): I) Depletion of stable RNAs using the Ribo-Zero rRNA removal kit II) RNA fragmentation by metal hydrolysis III)  Digestion of non native 5'-Mono/Di-phosphate ends by Terminator 5′-phosphate-dependent exonuclease IV)Conversion of 5′-triphosphates of primary transcript ends to monophosphates to enable adapter ligation V)Full RNA adapter ligation VI)cDNA synthesis by the usage of a loop adapter which hybridizes via a 3′-NNNNNNN-tail to the 3′-end of the RNA fragment VI)Amplification and Indexing by Adapter-specific primer