Investigation Title	Transcription profiling of wild type and sigma B mutant Liseteria monocytogenes to characterise the EGD-e sigma B regulon	
Comment[Submitted Name]	UGiessen_IMM_Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e sigma B regulon	
Experimental Design	dye_swap_design	co-expression_design	genetic_modification_design	transcription profiling by array	
Experimental Design Term Source REF	mo	mo	mo	EFO	
Comment[ArrayExpressReleaseDate]	2008-07-02	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-MEXP-1170	
Comment[MAGETAB TimeStamp_Version]	2010-08-09 21:08:17 Last Changed Rev: 13058	
Experimental Factor Name	time	genotype	
Experimental Factor Type	time	genotype	
Experimental Factor Term Source REF	
Person Last Name	Hain	
Person First Name	Torsten	
Person Mid Initials	
Person Email	Torsten.Hain@mikrobio.med.uni-giessen.de	
Person Phone	0049 641 99 46400	
Person Fax	0049 641 99 46409	
Person Address	Frankfurter Strasse 107, Giessen, Hessen, 35392, Germany	
Person Affiliation	Institute for Medical Microbiology	
Person Roles	submitter	
Person Roles Term Source REF	The MGED Ontology	
Quality Control Type	biological_replicate	dye_swap_quality_control	
Quality Control Term Source REF	The MGED Ontology	The MGED Ontology	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2008-07-02	
PubMed ID	
Publication DOI	
Publication Author List	
Publication Title	
Publication Status	
Publication Status Term Source REF	
Experiment Description	Briefly, over night cultures of L. monocytogenes EGD-e cells (wild-type and sigma B mutant) were grown in brain heart infusion (BHI) broth (Difco). Bacteria were diluted 1:50 in 20 ml fresh BHI and incubated using a 100 ml Erlenmeyer flask at 37 °C with shaking (180 rpm, Unitron [Infors]). Bacterial cells were harvested at OD600 1.0 (3 h), OD600 2.0 (4 h), OD600 2.5 (8 h) OD600 2.5 (16 h) using RNAprotect (Qiagen) according to manufacturer. 	
Protocol Name	P-MEXP-57899	P-MEXP-60549	P-MEXP-57900	P-MEXP-57901	P-MEXP-58053	P-MEXP-58060	
Protocol Type	nucleic_acid_extraction	labeling	labeling	hybridization	feature_extraction	bioassay_data_transformation	
Protocol Description	Total bacterial RNA was extracted using RNeasy Mini Kit (Qiagen) with a prior wash with SET buffer (50 mM NaCl, 5 mM EDTA and 30 mM Tris-HCl [pH 7.0]) containing 10% SDS, a pre-treatment at 37°C for 30 min at 350 rpm with 0.1 ml Tris-HCl [pH 6.5] containing 50 mg/ml lysozyme, 25 U of mutanolysin, 40 U of SUPERase, 0.2 mg of proteinase K (Ambion) and finally treated with 4 U of DNaseI (RNase free) (Ambion). The total RNA thus isolated was ethanol precipitated and quantified by absorbance at 260 nm and 280 nm and quality was analysed by Agilent 2100 Bioanalyzer. 	cDNA was generated and labeled with CyDye Cy5 using 3 µg of total RNA with CyScribe Post-Labeling Kit (GE Health Care). The cDNA thus generated was quantified by absorbance at 550 nm and 650 nm for Cy3 using ND-1000 Spectrophotometer (NanoDrop technologies).	cDNA was generated and labeled with CyDye Cy3 using 3 µg of total RNA with CyScribe Post-Labeling Kit (GE Health Care). The cDNA thus generated was quantified by absorbance at 550 nm and 650 nm for Cy3 using ND-1000 Spectrophotometer (NanoDrop technologies).	Hybridization was performed in ASP Base Hybridizer (GE Health Care) for 12 hours at 42°C (as per Type 7*[star] slide manufacturer’s instruction) using 10 pmol of Cy dye incorporated cDNA of wild-type and mutant, 50 µl of hybridization buffer (GE Health Care) and 100 µl of deionized Formamide (Ambion) adjusting the hybridization solution up to 200 µl with dd water (Ambion) per slide.	Hybridised Type 7*(star) slides were imaged with a Generation III Array Scanner (GE Health Care). The fluorescent signal intensities from each spot on the microarray were quantified using Spotfinder software (GE Health Care).	Data from image analysis were averaged over the duplicate spots present in one array, and then normalised in a two-step process; first, an intrachip normalisation was carried out in order to standardise signals from the Cy5 or the Cy3 channel. In a second step, we normalised over the entire series to bring the means to a common value. The intrachip normalisation was carried out using a robust local regression function (1) as implemented in the BIOCONDUCTOR package marrayNorm (http://www.bioconductor.org/). The parameters were: global local regression by the loess function, considering the inner 40% of data, i.e. those values where the log ratio was between the 30th and the 70th percentile of the distribution. For the second interchip normalisation the signals from the Cy3 channel of the first chip were taken as reference, and for all other signals the median of the difference of the logarithmised values was taken as a correcting factor. In a prior check of the correlation coefficient, low-signal values were excluded from the calculation when they deviated from linearity. For details, see (2). The performance of the normalisation procedure was controlled by inspecting scatter plots for all possible combinations of signals. Missing data from the cDNA microarray analyses were replaced by the corresponding mean gene expression values calculated from the biological replicates or eliminated when this information was not available.<br> <br> <br> (1)Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, and T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30:e15.<br> <br> (2) Beissbarth, T., K. Fellenberg, B. Brors, R. Arribas-Prat, J. Boer, N. C. Hauser, M. Scheideler, J. D. Hoheisel, G. Schutz, A. Poustka, and M. Vingron. 2000. Processing and quality control of DNA array hybridization data. Bioinformatics 16:1014-1022.	
Protocol Parameters	Amplification;Extracted product;	Amount of nucleic acid labeled;Amplification;Label used;	Amplification;Label used;Amount of nucleic acid labeled;	temperature;Volume;Chamber type;time;Quantity of label target used;	
Protocol Hardware					Generation III Array Scanner [Amersham]	
Protocol Software					Scanning software	
Protocol Contact	
Protocol Term Source REF		mo	mo		The MGED Ontology	The MGED Ontology	
SDRF File	E-MEXP-1170.sdrf.txt	
Term Source Name	mo		ncbitax	The MGED Ontology	ArrayExpress	The MGED Ontology	mo	EFO	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version