Illumina MiSeq paired end sequencing; Global gene transcription analysis in response to changes in chromosome supercoiling in Streptomyces coelicolor
Global gene transcription analysis in response to changes in chromosome supercoiling in Streptomyces coelicolor
RNA was isolated from S. coelicolor mycelia cultivated in 30 mL YEME/TSB liquid medium for 18 hours. RNA was extracted from vegetative hyphae of Streptomyces coelicolor growing for 18h in liquid YEME/TSB medium (30 mL). To induce supercoiling changes S. coelicolor culture was treated with novobiocin in final concentration 10 g/ml for 10 min (rapid chromosome relaxation). For long-term exposure to topological imbalance we used PS04 stain in which TopA level is controlled by thiostrepton addition (long-term high and low DNA supercoling in not induced and induced PS04 strain, respectively)  Mycelia were collected by centrifugation, frozen and stored at -70°C for subsequent RNA isolation medium: liquid YEME/TSB; 30 mL, 18 hours of growth, 30C, 180 rpm.  PS04 strain was indcuded with thiostrepton [1 µg/mL]. To indcude rapid chromosome relaxation M145 strain was treated with novobiocion [10 µg/mL] for 10 min before harvesting the culture. RNA was isolated using the procedure described previously by Moody et al. (Moody et al., 2013), digested with TURBO DNase I (Invitrogen, USA) RNA libraries were prepared for sequencing using standard Illumina protocols (NEBNext Ultra II Directional RNA Library Prep Kit). rRNA wesa removed using Ribo-Zero Ribosoma RNA Removal Kit (Illumina)
Experimental Factor: strain
PS04
Experimental Factor: phenotype
long-term increased DNA supercoiling