Investigation Title	Transcription profiling of MED8 N-truncation mutant, MED18 deletion mutant and MED20 deletion mutant yeast	
Comment[Submitted Name]	UMCU-MED-8	
Experimental Design	reference_design	dye_swap_design	genetic_modification_design	co-expression_design	transcription profiling by array	
Experimental Design Term Source REF	mo	mo	mo	mo	EFO	
Comment[ArrayExpressReleaseDate]	2008-07-09	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-MEXP-1175	
Comment[MAGETAB TimeStamp_Version]	2010-08-09 20:12:14 Last Changed Rev: 13058	
Experimental Factor Name	genotype	
Experimental Factor Type	genotype	
Experimental Factor Term Source REF	
Person Last Name	Sluiters	
Person First Name	Erik	
Person Mid Initials	
Person Email	e.c.sluiters@med.uu.nl	
Person Phone	+31-(0)30-2538348	
Person Fax	
Person Address	P.O. Box 85060	
Person Affiliation	Genomics Lab	
Person Roles	submitter	
Person Roles Term Source REF		
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2008-07-09	
PubMed ID	
Publication DOI	
Publication Author List	
Publication Title	
Publication Status	
Publication Status Term Source REF	
Experiment Description	expression profiles of MED8 N-truncation mutant, MED18 deletion mutant and MED20 deletion mutant against a wild type reference.	
Protocol Name	P-MEXP-74029	P-MEXP-74028	P-MEXP-74037	P-MEXP-74041	P-MEXP-74042	P-MEXP-74044	P-MEXP-25952	P-MEXP-28356	
Protocol Type	grow	specified_biomaterial_action	nucleic_acid_extraction	labeling	labeling	hybridization	feature_extraction	bioassay_data_transformation	
Protocol Description	S. cerevisiae BY4742 deletion mutants and the wild-type strains were cultured in SC Medium (Synthetic Complete; 4gr/2l Drop out mix 13.42gr/2l YNB(US biologicals) with 2% D-glucose)under agitation (250rpm), at 30°C. The cells were collected at midlog	Cells were spinned down at 3000rpm for 4 min. Pellets were taken up in liquid N2. Frozen cells (-80C) were resuspended in 500 ul Acid Phenol Chloroform (From Sigma, 5:1, pH 4.7). Immediately the same volume TES-buffer was added (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). Tubes were incubated for 1 hour in a thermomixer (65C, 1400RPM). Tubes were Spinned for 20 minutes at 14000 rpm at 4C. The water-phase was subjected to a second phenol chloroform extraction and subsequently by a Chloroform:Isoamyl-alcohol (25:1) extraction. RNA was precipitated with Sodium Acetate (NaAc, 3M, pH 5.2) and dissolved in water. RNA was subsequently cleaned using RNAeasy cleanup (Qiagen), including the DNase treatment.	Frozen cells (-80 „aC) were resuspended in 500 ul Acid Phenol Chloroform (From Sigma, 5:1, pH 4.7). Immediately the same volume TES-buffer was added (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). Tubes were incubated for 1 hour in a thermomixer (65C, 1400RPM). Tubes were Spinned for 20 minutes at 14000 rpm at 4C. The water-phase was subjected to a second phenol chloroform extraction and subsequently by a Chloroform:Isoamyl-alcohol (25:1) extraction. RNA was precipitated with Sodium Acetate (NaAc, 3M, pH 5.2) and dissolved in water. RNA was subsequently cleaned using RNAeasy cleanup (Qiagen), including the DNase treatment. RNA was frozen in liquid N2 and storen at -80C. 	RNA labeling with Cy-dyes · In dark micro centrifuge tube; dilute/concentrate required aRNA sample (x ng) to 8 µl. · Add 1 µl 0.5 M NaBicarbonate buffer, pH 9. Mix well. · Resuspend monofunctional NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): Quickly resuspend entire tube in 10 µl DMSO (Merck 8.02912.10). Add 1.25 µl per sample. Mix immediately and quickly. · Incubate 60 min at RT in the dark. · Add 4.5 µl 4 M hydroxylamine for quenching to prevent cross-coupling. · Incubate 15 min at RT in the dark. Cleanup using Clontech ChromaSpin-30 columns: 1. Upon removing a CHROMA SPIN Column from the protective plastic bag, invert it several times to resuspend the gel matrix completely. Before removing the top make sure that all the matrix is at the bottom by vigorously flicking the column downwards to remove matrix that is stuck to the top cap. 2. Holding the CHROMA SPIN Column upright, grasp the break-away end between your thumb and index finger and snap off. Place the end of the spin column into one of the 2-ml microcentrifuge (collection) tubes provided, and lift off the top cap. Save the top cap and the white-end cap. 3. Centrifuge at 700 g for 5 min (2700 RPM in Heraeus Megafuge 1.0 Sepatech Centrifuge, 1500 RPM in Eppendorf 5810 centrifuge (prog. 5)). After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration buffer from the column and re-establishes the matrix bed. Make sure that the matrix bed is flat. 4. Remove the spin column and collection tube from the centrifuge rotor, and discard the collection tube and column equilibration buffer. 5. Place the spin column into the second 2-ml microcentrifuge tube. Carefully and slowly apply the sample to the center of the gel bed's flat surface. Drop by drop! Do not allow any sample to flow along the inner wall of the column - this will completely mess up the sample. 6. Centrifuge at 700 g for 5 min. 7. Remove the spin column and collection tube from the rotor and detach them from each other. The purified sample is at the bottom of the collection tube. Immediately keep on ice and in the dark! Purified sample is about 15-30 µl. Bring sample up to 35 µl with water and take 5 µl eluate for spectrophotometry (190 to 750 nm range). Samples may be stored at -20	RNA labeling with Cy-dyes · In dark micro centrifuge tube; dilute/concentrate required aRNA sample (x ng) to 8 µl. · Add 1 µl 0.5 M NaBicarbonate buffer, pH 9. Mix well. · Resuspend monofunctional NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): Quickly resuspend entire tube in 10 µl DMSO (Merck 8.02912.10). Add 1.25 µl per sample. Mix immediately and quickly. · Incubate 60 min at RT in the dark. · Add 4.5 µl 4 M hydroxylamine for quenching to prevent cross-coupling. · Incubate 15 min at RT in the dark. Cleanup using Clontech ChromaSpin-30 columns: 1. Upon removing a CHROMA SPIN Column from the protective plastic bag, invert it several times to resuspend the gel matrix completely. Before removing the top make sure that all the matrix is at the bottom by vigorously flicking the column downwards to remove matrix that is stuck to the top cap. 2. Holding the CHROMA SPIN Column upright, grasp the break-away end between your thumb and index finger and snap off. Place the end of the spin column into one of the 2-ml microcentrifuge (collection) tubes provided, and lift off the top cap. Save the top cap and the white-end cap. 3. Centrifuge at 700 g for 5 min (2700 RPM in Heraeus Megafuge 1.0 Sepatech Centrifuge, 1500 RPM in Eppendorf 5810 centrifuge (prog. 5)). After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration buffer from the column and re-establishes the matrix bed. Make sure that the matrix bed is flat. 4. Remove the spin column and collection tube from the centrifuge rotor, and discard the collection tube and column equilibration buffer. 5. Place the spin column into the second 2-ml microcentrifuge tube. Carefully and slowly apply the sample to the center of the gel bed's flat surface. Drop by drop! Do not allow any sample to flow along the inner wall of the column - this will completely mess up the sample. 6. Centrifuge at 700 g for 5 min. 7. Remove the spin column and collection tube from the rotor and detach them from each other. The purified sample is at the bottom of the collection tube. Immediately keep on ice and in the dark! Purified sample is about 15-30 µl. Bring sample up to 35 µl with water and take 5 µl eluate for spectrophotometry (190 to 750 nm range). Samples may be stored at -20	PREHYBRIDIZATION: Make 100 ml prehybridization buffer (for a maximum of 4 slides) containing 5xSSC, 25% formamide (Merck Cat# 1.09684.10), 0.1% SDS and 1% bovine serum albumin (BSA; Sigma Cat# A-9418): 25 ml 20x SSC, 25 ml pure formamide, 49 ml ddH2O, 1 ml 10% SDS and 1 g BSA. After BSA has dissolved the solution becomes clear in about 5 minutes. Filter prehybridization buffer through a 0.22 micron syringe filter. Heat the prehybridization solution to 42C in a 100 ml Coplin Jar. Place slides to be analyzed into the preheated prehybridization buffer. Incubate for 45 minutes at 42C. Wash the slides by dipping 5 times in room temperature ddH2O (staining dish). Dip the slides 5 times in room temperature isopropanol (staining dish). Air dry in fume hood (15 ml tube rack upside down). Slides should be used immediately following prehybridization. HYBRIDIZATION: The hybridisation volume for a whole slide (25x60mm)is 500 ìl maximun volume of cy3 and cy5 combined targed is 250 ìl Optimum amount of target is 3000 ng fragmented cRNA per dye. Prepare 2x hybridisation buffer containing 50% formamide, 10xSSc and 0.2% SDS (2.5ml formamide; 2.5ml 20xSSC; 0.1ml 10% SDS). Immediately filter the hybridisation buffer through a 0.22micron filter (before SDS precipitates out of solution). Take 250 ìl of the 2x hybridisation buffer per slide. Add 5 ìlHerrring sperm DNA (stock 10ìg/ìl, sheared) per slide. Final concentration in 2x hybridisation buffer is 200ìg/ml. In case of all "non-yeast" arrays, also add 5ìl tRNA (stock 20ìg/ìl) per slide. Preheat to 42°C to overcome SDS precipitation. Combine 250ìl fragmented target with 250ìl 2x hybridisation buffer (preheated to 42°C) Heat at 95°C for 5 minutes. Spin at 12K for 2 minutes (RT). DO NOT place on ice after the 2 minute spin. Refer to the Agilent microarray hybridisation chamber user guide for in depth instructions on how to load the slides, assembly and disassembly of chambers as well as other helpful tips. Procure as many assembled chambers as needed to compleet your hybridisations. Disassemble chambers by turning the THUMBSCREW counterclockwise to loosen the CLAMP ASSEMBLY, slide off the CLAMP and remove the CHAMBER COVER. Load a clean GASKET SLIDE into the CHAMBER BASE with the LABEL facing up and aligned in the rectangular section of the CHAMBER BASE. Slowly dispense 490ìl of hybridisation solution onto the GASKET SLIDE in a "drag and dispense" manner as outlined in the Agilent microarray hybridisation chamber user guide (page 17). Flip the oligo array so that the back of the slide is facing up as you lower is gently onto the GASKET SLIDE. Quickly assess that the slides completely aligned. Re-align quickly if necessary. Do not move the CHAMBER BASE or sandwiched slides as this can cause leakage of the hybridisation mixture. Correctly plase the CHAMBER COVER onto the sandwiched slides and than slide on the CLAMP ASSEMBLY until it comes to a stopping point in the middle of the CHAMBER BASE and COVER pair. Tighten the THUMBSCREW by turning it clockwise until it is fully handtight. Hold the CHAMBER ASSEMBLY in your hand, VERTICALLY, and slowly rotate clockwise 2-3 times to allow the hybridisation solution to wet the GASKET. Inspect the sandwiched slides and note the bubble formation. A large mixing bubble should have formed. Small bubbles are acceptable if they move freely. Once all the chambers are fully assembled, load them into the hybridisation rotator rack. If you are not loading all the positions on the hybridisaion rotator rack, be sure to balance the loaded hybridisation chambers on the rack so that there are an equal number of empty positions on each of the four rows on the hybridisation rotator rack. Set the hybridisation rotator to rotate at 4 rpm. Hybridise at 42°C for 16-20 hours. WASHING THE HYBRIDIZED ARRAYS: Remove the slide from the hybridization chamber, taking care not to disturb the coverslip. Perform the following washes: (i) Low-stringency wash: Place the slides in a Coplin Jar containing 100 ml 1xSSC and 0.2% SDS (= 100 ml 1xSSC and 2 ml of 10% SDS). Gently remove the coverslip while the slide is in solution. LifterSlips are reusable and should be washed in 70% EtOH prior to use. Incubate for 4 minutes at room temperature. (ii) High-stringency wash: Place the slides in a Coplin Jar containing 100 ml 0.1xSSC and 0.2% SDS (= 10 ml 1xSSC, 90 ml water and 2 ml of 10% SDS). Incubate for 4 minutes at room temperature. Wash the slide finally in 100 ml 0.1xSSC to remove particles of SDS. Incubate for 4 minutes at room temperature. Allow the slides to dry by centrifuge 1 minute 500 rpm (do this immediately after the third wash).	Scanning of slides using the Agilent G2565AA DNA microarray scanner and the Agilent Feature Extraction software version 7.5 <br> PMT 30, laser power 100	All data was normalized using the marray package (1.5.8) from Bioconductor, resulting in the following output:<br> <br> * Flag_Cy3, Flag_Cy5 <br>   User flags for bad spots<br> <br> * Signal Norm_Cy3, Signal Norm_Cy5<br>   Normalized values for the Cy3 and Cy5 channels. These values were derived by performing a lowess normalization per print-tip on genes (with a window size of 0.4) on the background substracted 'Signal Mean' columns from the raw data file (i.e. Signal Mean_Cy3 - Background Mean_Cy3). Spots with negative values after background substraction were omitted from subsequent analysis.<br> <br> * M<br>   The M-transformed values of the Signal Norm_Cy3 and Signal Norm_Cy5 rows.<br>   M = log2(Signal Norm_Cy5 / Signal Norm_Cy3)<br>   <br> * A<br>   The A-transformed values of the Signal Norm_Cy3 and Signal Norm_Cy5 rows.<br>   A = 1/2 * log2(Signal Norm_Cy5 * Signal Norm_Cy3)	
Protocol Parameters			Amplification;Extracted product;	Label used;Amplification;	Label used;Amplification;	Chamber type;temperature;Quantity of label target used;	
Protocol Hardware							G2565AA DNA microarray scanner [Agilent]	
Protocol Software							Feature Extraction Software [Agilent]	
Protocol Contact	
Protocol Term Source REF							The MGED Ontology	The MGED Ontology	
SDRF File	E-MEXP-1175.sdrf.txt	
Term Source Name	mo		ncbitax	The MGED Ontology	ArrayExpress	mo	EFO	The MGED Ontology	
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://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version