title: CrsL KD 3 sta
organism: Mycolicibacterium smegmatis MC2 155
description: Protocols: 50 ml exponential phase cells and 25 ml stationary phase cells were pelleted by centrifugation and frozen at -70°C. Mycobacterium smegmatis mc2 155 cells (negative control (LK2261), carD/MSMEG_6077 knockdown strain (LK3301) and crsL/MSMEG_5890 knockdown strain (LK3302)) were inoculated to OD600 0.1 and grown at 37°C in Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80. For exponential phase cells, knockdowns were induced with anhydrotetracycline (100 ng/mL) after 3 h of OD600 0.1 and the strains were cultivated for another 3 h and harvested (OD600 ~0.5). For stationary phase cells, knockdowns were induced with anhydrotetracycline (100 ng/mL) after 8 h of OD600 0.1 and the strains were cultivated for another 16 h to reach early stationary phase (OD600 ~2.5). For unbiased quantification, RNA spike-in were added to each sample according to the following rule: 15 ng of RNA spike-in were added per 30 ml culture of OD600~0.5. Cell pellets were vortexed in 240 μl TE (10 mM Tris, pH 8.0; 1 mM EDTA), 60 μl LETS (50 mM Tris–HCl pH 8.0, 500 mM LiCl, 50 mM EDTA pH 8.0, 5% SDS), 600 μl acidic phenol: chloroform (1:1) were added, sonicated for 30 seconds, centrifuged and the aqueous phase was transferred. 600 μl acidic phenol: chloroform (1:1) was added, the samples were vortexed and again, the aqueous phase was transferred. This step was repeated once more. Then, the RNA was precipitated with ethanol, dissolved in double distilled water, DNase treated (TURBO DNA-free Kit, Ambion) and approximately 1 μg of DNase treated RNA was ribodepleted (riboPOOL Kit Pan-Actinobacteria, siTOOLs). The sample integrity was checked using Agilent 2100 Bioanalyzer Pico Chip. The ribodepleted RNA sample (20-100 ng) was used for library construction according to the NEXTFLEX® Rapid Directional RNA-Seq Kit. The libraries were checked using Agilent 2100 Bioanalyzer Nano Chip.