title: Illumina HiSeq 2000 sequencing; RNA-seq of wild type Mycobacterium smegmatis mc2 155 (exponential and stationary phase) and Ms1 deletion strain (exponential and stationary phase)
design: RNA-seq of wild type Mycobacterium smegmatis mc2 155 (exponential and stationary phase) and Ms1 deletion strain (exponential and stationary phase)
library_construction_protocol: 25 ml cells from the indicated growth phases were quickly cooled on ice, pelleted and immediately frozen.  M. smegmatis mc2 155 strains were grown at 37°C in Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80 and harvested in exponential phase (OD600 ∼0.5) or after 24 hours in stationary phase (OD600 ∼2.5–3). In Ms1 deletion strain, Ms1 gene is replaced by hygromycine antibiotic resistance (LK1611). The pellet was then suspended in 240 ul TE (pH 8.0) plus 60 ul lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM LiCl, 50 mM EDTA pH 8.0, 5% SDS) and 600 ul acidic phenol (pH~3):chloroform (1:1). Lysates were sonicated in a fume hood, centrifuged, the aqueous phase extracted two more times with acidic phenol (pH~3): chloroform and precipitated with ethanol. RNA was dissolved in water and DNase treated (TURBO DNA-free Kit, Ambion) two times. Five micrograms of total RNA were rRNA depleted with RiboZero Magnetic Kit, Gram positive Bacteria (Epicentre) and strand specific libraries prepared with Illumina compatible NEXTflex Rapid Directional RNA-Seq Kit (Bioo Scientific) according to manufactuer´s instructions.
attributes.tag: Experimental Factor: genotype
attributes.value: Ms1 deletion
attributes.tag: Experimental Factor: growth condition
attributes.value: logarithmic phase culture