strain: MG1363 genotype/variation: IS905 integration in llmg_1239
Treatment protocol
Cultures were grown to mid-exponential (OD600 of around 0,3 or 0,6) phase.
Growth protocol
Cultures were grown in closed vessles at 30°C in rich M17 medium containing 1% cellobiose as a carbon source.
Extracted molecule
total RNA
Extraction protocol
As described in Marredy et al., 2011. Cells were harvested immediately upon reaching the required optical density, snap frozen in liquid nitrogen and stored at -80°C. Cells were lysed by bead beating and chromosomal DNA was removed by Macaloid suspension (Bentone MA, Hightstown, NJ). Proteins were removed from the cell lysate by chloroform/phenol extraction and total RNA was isolated from the water phase using the Pure RNA Isolation Kit (Roche Molecular Biochemicals, Mannheim, Germany).
Label
Cy3
Label protocol
cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) and allyl-modified dUTPs to allow indirect Cy-3/Cy-5 labelling according to manufacturers instructions (Amersham Biosciences, Piscataway, NJ).
Hybridization protocol
Superamin glass slides (ArrayIt, Sunnyvale, CA) were spotted with duplicates of approximately 2500 ORF amplicons of L. lactis subsp. MG1363. Hybridization was performed in Ambion Slidehyb #1 hybridisation buffer (Ambion Biosystems, Foster City, CA) for 16 h at 45°C in a microarray hybridization incubator ISO20 (Grant Boekel, Cambridgeshire, UK). Slides were washed in 2x SSC (+0.5 % SDS), 1x SSC (+0.25% SDS) and 1x SSC (+0.1% SDS), prior to drying and scanning.
Scan protocol
Slides were scanned using a GenePix Autoloader 4200AL scanner (Molecular Devices Corporation, Sunnyvale, CA)
Description
L. lactis MG1363ΔcelBΔptcCΔ0963 llmg_1239::IS905 biological replicate 2, labeled with Cy3
Data processing
Data analysis was carried out using ArrayPro 4.5 (Media Cybernetics, Silver Spring, MD) for image analysis and processed using a lowess normalization assuming that at least 50% of the genes will show no change in expression. Data was compiled from two anaerobic cultures each with a dye-swap replicate, providing two biological repeats within four technical repeats. Raw signals were normalized using lowess normalizaton and scaled using Trimmed Median Mean (TMM)
