For transcriptome analysis C. glutamicum MB001 and C1 cells were grown in 5 ml BHI (Brain Heart Infusion, Difco) for about 6 hours at 30°C. A second precultivation was performed in CGXII minimal medium containing 2% (w/v) glucose as carbon source. The main cultures were inoculated to an OD of 1 in CGXII minimal medium with 2% (w/v) glucose. At an OD of 5 the cells were harvested by centrifugation (4120 x g, 10 min and 4 °C). The cell pellet was subsequently frozen in liquid nitrogen and stored at -70°C.
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott, (2004) Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol Microbiol 54: 420-438.)
Label
Cy5
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm. 2003. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl. Environm. Microbiol. 69:2521-2532 and in Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott. 2004. Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol. Microbiol. 54: 420-438.
For transcriptome analysis C. glutamicum MB001 and C1 cells were grown in 5 ml BHI (Brain Heart Infusion, Difco) for about 6 hours at 30°C. A second precultivation was performed in CGXII minimal medium containing 2% (w/v) glucose as carbon source. The main cultures were inoculated to an OD of 1 in CGXII minimal medium with 2% (w/v) glucose. At an OD of 5 the cells were harvested by centrifugation (4120 x g, 10 min and 4 °C). The cell pellet was subsequently frozen in liquid nitrogen and stored at -70°C.
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott, (2004) Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol Microbiol 54: 420-438.)
Label
Cy3
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm. 2003. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl. Environm. Microbiol. 69:2521-2532 and in Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott. 2004. Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol. Microbiol. 54: 420-438.
Hybridization protocol
Purified cDNA samples to be compared were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent’s Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
Scan protocol
Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 6.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0).
Data processing
For ratio calculation and ratio normalization, GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).
