NextSeq 500 paired end sequencing; RNA-seq of DNA gyrase expressing Thermococcus kodakarensis
RNA-seq of DNA gyrase expressing Thermococcus kodakarensis
For preparation of samples for RNA sequencing, 25 ml of Ravot medium was inoculated at 1/100 dilution with fresh T. kodakarensis preculture.  Total RNA was extracted from 20 ml of exponentially growing cultures (6 hours of culture, approximatively 2x109 cells/ml) using a NucleoSpin RNA set for NucleoZOL (Macherey Nagel). DNA was eliminated from samples using a TURBO DNA-free kit following the manufacturers protocol (Ambion). For each strain, samples were prepared from four independent cultures. Total RNA quality was assessed on an Agilent Bioanalyzer 2100, using RNA 6000 pico kit (Agilent Technologies).  Directional RNA-Seq Libraries were constructed using the TruSeq Stranded Total RNA library prep kit, with bacteria Ribo-Zero reagents (Illumina), following the manufacturerâ€™s instructions; 500ng of total RNA were used. After the Ribo-Zero step, the samples were checked on the Agilent Bioanalyzer for proper rRNA depletion. Final libraries quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit.
Experimental Factor: phenotype
expression of inactive T. maritima DNA gyrase