Comment[ArrayExpressAccession]	E-MTAB-2127
Investigation Title	Comparison of gene expression in S.aureus strains with different triclosan sensitivities
Comment[Submitted Name]	Comparison of gene expression in S.aureus strains with different triclosan sensitivities
Experiment Description	Difference in sensitivity for the biocide triclosan has been observed in S.aureus. This may rely on changes in gene expression between strains. The present project investigates such changes in in vivo generated mutants and in clinical isolates.
Public Release Date	2015-04-30
Comment[AEExperimentType]	transcription profiling by array
Experimental Design	strain or line design
Experimental Design Term Source REF	Experimental Factor Ontology
Experimental Factor Name	triclosan sensitivity
Experimental Factor Type	response to drug
Person Last Name	Grandgirard
Person First Name	Denis
Person Email	denis.grandgirard@ifik.unibe.ch
Person Phone	+41 31 632 4949
Person Address	Friedbuelstrasse 51, 3010 Bern, Switzerland
Person Affiliation	University of Bern, Institute for Infectious Diseases
Person Roles	submitter
Quality Control Type	biological replicate
Quality Control Term Source REF	Experimental Factor Ontology
Replicate Type	biological replicate
Normalization Type	stagewise quantile normalization
Publication Author List	Denis Grandgirard, Leonardo Furi, Maria Laura Ciusa, Lucilla Baldassarri, Daniel R. Knight, Ian Morrissey, Carlo R Largiader, Stephen L Leib, Marco R Oggioni
Publication Title	Mutations upstream of fabI in triclosan resistant Staphylococcus aureus are associated with elevated level of fabI gene expression
Publication Status	submitted
Protocol Name	P-MTAB-36387	P-MTAB-36388	P-MTAB-36389	P-MTAB-36390	P-MTAB-36391	P-MTAB-36392	P-MTAB-36393
Protocol Type	growth condition design	extraction	labelling	hybridization	scanning	normalization	data transformation
Protocol Description	S. aureus strains were grown overnight in 10 ml tryptic soy broth (TSB) at 37°C at 80 rpm. The culture were diluted 1:100 in pre-warmed TSB and grown to logarithmic phase (OD570 =0.6). 2 ml of the culture (1-5 x 10^8 colony forming units) were harvested in 4 ml of RNAprotect® reagent (Qiagen), incubated for 5 min at room temperature and centrifuged for 10 min at 5000 x g. The pellet was then processed directly for RNA extraction or stored at -80°C for later processing	Bacterial pellets were resuspended in 200 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8) containing 50 U recombinant lysostaphin (Sigma) and incubated for 10 min at 37°C, 600 rpm. 1 ml of hot Qiazol (Qiagen) added and bacteria, incubated for 5 min at room temperature. Bacteria were further disrupted by vibration with 50 mg of acid-washed glass beads (Sigma) using a Mickle Vibratory Tissue Disintegrator (Mickle Laboratory Engineering) at maximum speed. Samples were extracted with 200 µl chloroform for 3 min at room temperature. Total RNA was then extracted using RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Contaminating DNA was removed using DNA-free™ Kit (Applied Biosystems) . RNA concentration and purity (OD 260/280) were determined by Nanodrop® ND-1000 spectrophotometer (Thermo Scientific).	Isolated, unamplified RNA was labelled with Cy5, using ULS™ Labeling Kit for CombiMatrix arrays (Kreatech Biotechnology), according to the manufacturer’s instructions (http://www.kreatech.com/uploads/pics/4-40_Combimatrix_Labeling_v2.0.pdf) . RNA was finally fragmented with RNA Fragmentation Reagents (Ambion®)	12K Customarrays were hybridized with 2-3 µg of labelled, fragmented RNA, according to information provided by the manufacturer (Customarray/Combimatrix Incorporated, http://customarrayinc.com/products_arrays.htm)	Microarrays were scanned using the Packard ScanArray4000 array scanner and software (ScanArray, version 3.1, Packard BioChip Technologies). All arrays were scanned with incremental laser power (LP) from 15 to 100%. Data were extracted with Microarray Imager software (version5.8.0, Combi Matrix) and spot intensity expressed as median intensity	Non-specific binding was determined from fluorescence values of all non-specific probes. The cut-off for specific binding was set as the upper 95% confidence interval of the mean signal intensity of the non-specific probes. For each comparison, probes were excluded when the mean values for both strains to be compared were under the determined cut-off. For comparisons involving in vitro generated mutants derived from plasmid-free strains RN4220 or ATCC6538, analysis was performed using only probes sets gathered from the genomes of S. aureus TW20 and Mu50. The fluorescence values of the filtered probes were log2 transformed. For each set of comparison, stage-wise quantile normalization was performed, using a script written in the statistical computing environment of R (R Development Core Team, 2011), according to Deshmukh et al. (Deshmukh, S. R. and S. G. Purohit. 2007. Microarray Data: Statistical Analysis Using R. Alpha Science International Ltd., Oxford U.K.)	Scanning data with approaching median fluorescence intensity (MFI) for each experiment were chosen for analysis. Fluorescence values of spots with maximal intensity (signal saturation) at the chosen laser power intensity (chosen LP) were extrapolated by linear regression, using values gathered from the two next lower laser intensities.
Protocol Parameters				amount of RNA(ug)			Chosen Laser Intensity (LP)
Protocol Term Source REF	Experimental Factor Ontology	Experimental Factor Ontology	Experimental Factor Ontology	Experimental Factor Ontology	Experimental Factor Ontology	Experimental Factor Ontology	Experimental Factor Ontology
Protocol Hardware					Packard ScanArray4000
Protocol Software					ScanArray, version 3.1; Microarray Imager software (version5.8.0, Combi Matrix)
Term Source Name	Experimental Factor Ontology	National Cancer Institute Thesaurus
Term Source File	http://bioportal.bioontology.org/ontologies/39885	http://ncich.nci.nih.gov/
Term Source Version	2.38
Comment[AdditionalFile:txt]	readme.txt	ATCC25923vsQBR1052_normalised.txt	ATCC25923vsQBR12363_normalised.txt	ATCC25923vsQBR1889_normalised.txt	ATCC25923vsQBR1969_normalised.txt	ATCC6538vsMO052_normalised.txt	RN4220vsMO034_normalised.txt	RN4220vsMO035_normalised.txt	RN4220vsMO036_normalised.txt
Comment[AdditionalFile:pdf]	normalisation_data_relationship.pdf
SDRF File	E-MTAB-2127.sdrf.txt
PubMed ID	25924916
Publication DOI	10.1186/s12864-015-1544-y