Comment[ArrayExpressAccession] E-MEXP-3319 Investigation Title Impact of RelRS and CiaRH deletion in transcription profiling in a wild-type background Comment[Submitted Name] Impact of RelRS and CiaRH deletion in transcription profiling in a wild-type background Comment[AEExperimentDisplayName] Impact of RelRS and CiaRH deletion in transcription profiling in a wild-type background Comment[MIAMExpressLogin] vdacunha Comment[MIAMExpressSubmissionID] 7718 Experiment Description Impact of two TCS RelRS and CiaRH deletion in transcription profiling in a wild-type background Experimental Design co-expression_design genetic_modification_design dye_swap_design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name GENOTYPE Experimental Factor Type genotype Person Last Name DA CUNHA Person First Name Violette Person Email vdacunha@pasteur.fr Person Phone 7604 Person Affiliation Institut Pasteur Person Address Genomes et Génétique, 25-28 rue du Docteur Roux, PARIS, PARIS, 75015, France Person Roles submitter Person Roles Term Source REF MGED Ontology Quality Control Type Public Release Date 2014-05-25 Comment[ArrayExpressSubmissionDate] 2011-07-29 12:36:33 Publication Status not yet submitted Protocol Name P-MTAB-22298 P-MTAB-22299 P-MTAB-22300 P-MTAB-22301 P-MTAB-22302 P-MTAB-22303 Protocol Description No background was subtracted. Data normalization and differential analysis were conducted using the R software (http://www.r-project.org). A global intensity-dependent normalization using the LOESS procedure (Yang et al,2002) was performed on a slide-by-slide basis (BioConductor package marray; http://www.bioconductor.org/packages/bioc/html/marray.html) to correct the dye bias.

To determine differentially expressed genes, we performed a paired t test using the commun method, together with the Benjamini and Yekutieli p-value correction method. Cy3 and Cy5 targets quantity were normalized at 150 pmol, mixed and thereafter concentrated using Microcon YM-30 (Millipore). Samples volumes were adjusted to 20µl in nuclease-free water, and used for Agilent hybridization protocol(Two color microarray-based gene expression analysis protocol session Hybridization)
(Parameters: Chamber type = OTHER: Agilent technology chambers, Quantity of label target used = 300, Mass unit = Nano gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 65) Slides are placed in an Axon 4200AL scanner connected to the GenePix Pro 6.0 image acquisition and analysis software package. Slides are scanned at 635nm and 532nm wavelength (respectively for Cy5 and Cy3). An initial pre-scan of the slide is performed to determine success of hybridisation and to allow the user to define the boundaries of the array features. PMT gain settings are adjusted at this step to ensure that the overall red-to-green ratio is similar. The slides are scaned and the resulting images are saved as a .TIF file for analysis and feature extraction using GenePix Pro.
(Parameters: Scanning hardware = OTHER: Axon 4200AL, Scanning software = GenePix Pro [Axon Instruments]) S. agalactiae strains were cultured over-night at 37C. Then they were diluted to OD=0.05 and grow it until the desired OD.- Harvest 25 ml of the bacterial cells by centrifugation at 5000 g for 5 min at 4 degrees C. Discard the supernatant. You can use the pellet immediately or freeze it into liquid nitrogen. - Prepare 500 µl of acidic phenol pH 4.5 and 0.4 g of glass beads (200-300 microns Sigma) into a Sarsted tube. Resuspend the bacterial pellet in 400 µl resuspension buffer (1/2 volume glucose 20% + 1/2 volume Tris-HCl 25 mM pH7.6 EDTA 10mM) and 60 µl EDTA 0.5 M. Vortex and transfer the suspension into the tube containing acidic phenol and beads. - Disrupt the cells in a FastPrep apparatus (Bio101) with the indicating parameters (Speed: 6, Time: 30 s). Perform this cycle twice with 1 min at 4 degrees C between each cycle. Centrifuge 5 min at 13,000 rpm at 4 degrees C. Transfer the aqueous phase in a sterile Eppendorf tube. - Add 1 ml Trizol (Gibco). Mix thoroughly and incubate at room temperature for 5-10 min. Add 100 µl chloroform/isoamylic alcohol (24/1 v/v). Mix vigorously and incubate for 1 min at room temperature. - Centrifuge 5 min at 13,000 rpm at 4 degrees C. Transfer the aqueous phase in a sterile tube. Add 200 µl chloroform/isoamylic alcohol (24/1 v/v). Mix gently by pipetting and incubate 5 min at room temperature. - Centrifuge 5 min at 13,000 rpm at 4 degrees C. Transfer the aqueous phase in a new sterile tube. Add 500 µl isopropanol (room temperature). Mix by inverting the tube. Incubate 30 min on ice. - Centrifuge 15 min at 13,000 rpm at 4 degrees C. Discard the supernatant and resuspend the pellet in 100 µl DEPC water. Add 10 µl NaAcetate (pH 5.2) and 250 µm EtOH p.a. and incubate at -20 degrees C for 2h to ON. - Centrifuge 15 min at 13,000 rpm at 4 degrees C. Discard the supernatant and wash the pellet with 1 ml of ice-cold ethanol 70%. Centrifuge 5 min at 13,000 rpm at 4 degrees C. - Remove the supernatant and dry the pellet. Resuspend pellet in 50 µl DEPC water and store RNA at -80 degrees C.

The RNA quality is tested on RNA Nano Chips with the Agilent 2100 bioanalyzer.
(Parameters: Extracted product = total_RNA, Amplification = none) S. agalactiae mutant and wildtype were cultured in TH at 37C. S. agalactiae was harvested for RNA isolation at the exponential phase (OD 0.3-0.4). 25 ml of the bacterial cells at the desired OD were centrifugated at 5000 g for 5 min at 4 degrees C. Discard the supernatant. And the pellet were immediately freezed and stored at -80°C to prevent RNA degradation
(Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = TH) RNA samples (5µg) were indirectly labelled using Superscript Indirect cDNA labeling system kit (Invitrogen) with a mixture of random hexamer (pdN6), according to the contitions recommanded by the manufacturer Targets quality and concentration were determined by spectroscopy at 260nm, 280nm and 650nm.
(Parameters: Amplification = none, Mass unit = Micro gram) Protocol Type bioassay_data_transformation hybridization image_acquisition nucleic_acid_extraction grow labeling Protocol Term Source REF MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology Term Source Name MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php SDRF File E-MEXP-3319.sdrf.txt