title: Illumina MiSeq paired end sequencing; The guanidinobutyrase GbuA is essential for the alkylquinolone-regulated pyocyanin production during parasitic growth of Pseudomonas aeruginosa in co-culture with Aeromonas hydrophila
design: The guanidinobutyrase GbuA is essential for the alkylquinolone-regulated pyocyanin production during parasitic growth of Pseudomonas aeruginosa in co-culture with Aeromonas hydrophila
library_construction_protocol: Preculture cells of  Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PAO1∆gbuA  were washed and used to inoculate main cultures in 4 ml medium B (50 mM Hepes (pH 7), 0.5 mM MgSO4, 14 mM KCl, 7.2 mM NaCl, 5 mM NH4Cl. After autoclaving, the medium was complemented with 0.01 mM CaCl2, 0.15 mM Na-K-phosphate buffer (pH 7; 0.105 mM K2HPO4, 0.045 mM NaH2PO4) and the trace element solution SL10 (Widdel et al., 1983))  containing 20 mM succinate and 100 μM 4-GB in 15 ml test tubes in triplicates with an OD600 of 0.001. After incubation for 11 h at 37°C in at 200 r.p.m. in a rotary shaker, the respective triplicates were combined, harvested by centrifugation and resuspended in medium B without complementing solutions. RNA was extracted with the innuPREP RNA Minikit from Analytik Jena (Jena, Germany) according to the manufacturer’s instructions. Residual DNA was removed by digestion with 10 U RNase-free DNase I (Thermo scientific) for 1 h in the presence of RiboLock RNase inhibitor (Thermo scientific). After DNA digestion, the RNA was again purified with the same kit. RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Boblingen, Germany). Ribosomal RNA molecules were removed from total RNA with the Ribo-Zero rRNA Removal Kit (Illumina, San Diega, USA) and removal of rRNA was checked with the Agilent RNA Pico 6000 Kit on an Agilent 2100 Bioanalyzer.
attributes.tag: Experimental Factor: gbuA
attributes.value: genotype