title: Illumina MiSeq paired end sequencing; Whole track RNAseq of the deletion mutant ∆cgt in Actinoplanes sp. SE50/110 during growth phase and during transition into stationary phase
design: Whole track RNAseq of the deletion mutant ∆cgt in Actinoplanes sp. SE50/110 during growth phase and during transition into stationary phase
library_construction_protocol: For transcriptome analysis, 2x 1 mL samples from Actinoplanes culture were taken during exponential growth phase, separated from the supernatant by centrifugation (10 s) and snap-frozen in liquid nitrogen. Pellets were stored at -80 °C until further processing. Cultivation was performed in 250 mL Corning® Erlenmeyer baffled cell culture flasks at 28 °C and 140 rpm for seven days in maltose minimal medium (72.06 g?L-1 maltose·1H2O, 5 g?L-1 (NH4)2SO4, 0.184 g?L-1 FeCl2·4H2O, 5.7 g?L-1 Na3C6H5O7·2H2O, 1 g?L-1 MgCl2·6H2O, 2 g?L-1 CaCl2·2H2O, trace elements (final concentration: 1 µM CuCl2, 50 µM ZnCl2, 7.5 µM MnCl2 dissolved in 1 M HCl) and phosphate buffer consisting of 5 g?L-1 each K2HPO4 and KH2PO4 in aqua distilled). The medium was prepared and filter sterilized following the protocol of Wendler et al. (2013). For inoculation of 50 mL medium, 1 mL spore solution of an OD = 3-5 was used. Cell dry weights were determined like described by Wolf et al. (2017). The supernatant was stored for later analysis at -20 °C. For RNA isolation, frozen cell pellets were resuspended in 500 µL LB-buffer (NucleoSpin® RNA Plus, Macherey-Nagel, Düren, Germany) and transferred to 2 mL lysing matrix tubes (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, California, USA). Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) for three times 20 s at speed setting 6.5 and 5 min on ice in between. Subsequently, the cell suspension was centrifuged for 5 min at 13,000 g and 4 °C. The supernatant was used for RNA extraction using the NucleoSpin® RNA Plus Kit in combination with rDNase Set (Macherey-Nagel, Düren, Germany) for an on-column DNA digestion. After clean-up and elution according to manufacturer's protocol, the DNA-digestion was repeated in-solution and the sample cleaned up again by use of the same kit. Residual DNA was tested negatively with two primer pairs binding to genomic DNA of Actinoplanes sp. SE50/110 and amplifying small fragments at round about 200-300 nt. The quantity of RNA was analyzed with the NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany). RNA of triplicates of the wildtype and the deletion mutant was isolated from growing cultures and pooled equimolar separately for the wildtype and the deletion mutant (total amount of 2.5 µg RNA each in 26 µL RNase-free water). rRNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina, San Diego, USA). Successful depletion of rRNA was verified by an Agilent RNA 6000 Pico chip in the Bioanalyzer (Agilent, Böblingen, Germany). cDNA libraries were prepared following protocols from Pfeifer-Sancar et al. (2013) and Irla et al. (2015) by use of the TruSeq stranded mRNA kit (Illumina, San Diego, CA, USA). The libraries were quantified by a DNA High Sensitivity Assay chip in the Bioanalyzer (Agilent, Böblingen, Germany).
attributes.tag: Experimental Factor: genotype
attributes.value: wild type
attributes.tag: Experimental Factor: growth condition
attributes.value: growth phase