GSM1368033: Cip 1; Pseudomonas putida DOT-T1E; RNA-Seq
Single colonies of P. putida strain DOT-T1E were grown overnight in Luria–Bertani (LB) medium at 30°C. Overnight cultures were diluted to a starting OD600 of 0.01 in the same medium and 50 ml aliquots were disposed in separate 250 ml Erlenmeyer flasks and incubated with shaking at 200 rpm. When cultures reached exponential phase (0.5 at OD600), antibiotics were added at sub-lethal concentrations to the culture medium to reach a final concentration of 1 µg/ml kanamycin, 300 µg/ml ampicillin, 150 µg/ml chloramphenicol, 4 µg/ml tetracycline, 0.5 µg/ml ciprofloxacin, 300 µg/ml spectinomycin, 500 µg/ml rifampicin and 2 µg/ml gentamicin. Then cultures were incubated under the same conditions for one hour more. Cells were harvested by centrifugation at 8000 g for 10 min and suspended immediately in stop solution (95% (v/v) ethanol, 5% (v/v) phenol) and pelleted by centrifugation. After that, total RNA was extracted with Trizol (Invitrogen). Removal of DNA was carried out by treatment with DNase I (Fermentas) in combination with the RNase inhibitor RiboLock (Fermentas). The integrity and quality of total RNA was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM1368033