title: Illumina MiSeq paired end sequencing; Global gene transcription analysis in response to changes in chromosome supercoiling in Streptomyces coelicolor
design: Global gene transcription analysis in response to changes in chromosome supercoiling in Streptomyces coelicolor
library_construction_protocol: 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)
attributes.tag: Experimental Factor: strain
attributes.value: PS04
attributes.tag: Experimental Factor: phenotype
attributes.value: long-term increased DNA supercoiling