2025-11-01
ENA first public
ERC000011
ENA-CHECKLIST
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Czech Republic Department of Microbial Genetics and Gene Expression, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
INSDC center name
public
INSDC status
ERS18293800
SRA accession
E-MTAB-13812:CrsL KD 2 sta
Submitter Id
ArrayExpress
broker name
not collected
collection date
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.
description
PLJR962_crsL knockdown
genotype
not collected
geographic location (country and/or sea)
stationary phase
growth stage
not applicable
isolate
Mycolicibacterium smegmatis
organism
Mycolicibacterium smegmatis
organism
whole organism
organism part
Mycolicibacterium smegmatis
scientific_name
MC2 155
strain
CrsL KD 2 sta
title