ONTOLOGY SOURCE REFERENCE
Term Source Name	"CHEBI"	"BTO"	"NCBITAXON"	"UBERON"	
Term Source File	"http://data.bioontology.org/ontologies/CHEBI"	"http://data.bioontology.org/ontologies/BTO"	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/UBERON"	
Term Source Version	"78"	"20"	"2"	"191"	
Term Source Description	"Chemical Entities of Biological Interest Ontology"	"BRENDA Tissue and Enzyme Source Ontology"	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"Uber Anatomy Ontology"	
INVESTIGATION
Investigation Identifier	"1406042467633"
Investigation Title	""
Investigation Description	""
Investigation Submission Date	""
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Comment [Created with configuration]	""
Comment [Last Opened With Configuration]	""
Comment[Created With Configuration]	""
Comment[Last Opened With Configuration]	""
INVESTIGATION PUBLICATIONS
Investigation PubMed ID	""
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INVESTIGATION CONTACTS
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STUDY
Study Identifier	"E-GEOD-10621"
Study Title	"Transcription profiling of human ciliary smooth muscle cells grown culture with 200 nM of either a prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. Culture conditions included 100 nM indomethacin."
Study Description	"Changes of genome-wide mRNA transcription levels of human ciliary smooth muscle (hCSM) cells were determined by treating hCSM cells in culture with 200 nM of either an prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. This was followed by competitive hybridization of fluorescent Cy3 or Cy5 labeled cRNA probes derived from the treated versus untreated control total RNA samples onto an Agilent Human Whole Genome Expression oligonucleotide microarray. Log 2 (LN) of the intra-slide ratios (RATIO, PRE_VALUE) of treated versus untreated samples was reported as VALUE in the sample files. Experiment Overall Design: Human ciliary smooth muscle (hCSM) cells were isolated from a female donor, received from the San Diego Eye Bank® (San Diego, CA) and cultured in DMEM with 10% fetal bovine serum and 0.5% penicillin/streptomycin according to the method reported previously by Woldemussie et al. For transcriptome analysis, cells were used for experiments starting at passage 4. Three independent experiments at consecutive cell passages were performed. For each experiment 0.6 x 10^6 cells were plated onto 15 cm dishes in MEM D-Valine medium (PromoCell, Germany) supplemented with 10% FCS, L-glutamine and antibiotics/antimycotics. Cells were grown to ~80% confluency for 6 days, followed by starvation in absence of FCS and presence of 100 nM of the cyclooxygenase inhibitor indomethacin, 20 µg/ml fatty acid-free BSA and 4 µg/ml transferrin for 17 hours. Cells were then exposed for 6 hours to 200 nM of either the EP2 agonist AGN210937 or the EP4 agonist AGN202280 (Allergan, Irvine, CA) or the corresponding amount of DMSO in the absence of FCS and presence of transferrin. Total RNA was isolated using a RNA isolation kit (RNAqueous; Ambion, USA) which was followed by DNase I treatment (Turbo DNA-free; Ambion) and purification, according to the manufacturer's protocols. RNA was quantitated using a spectrophotometer ND-1000 (Nanodrop Technologies, USA). RNA quality was assessed regarding purity and stability using a Bioanalyser 2100 (Agilent Technologies, USA). Extracted total RNA aliquots were snap-frozen in liquid nitrogen and stored at -80°C for single use. Total RNA was linearly amplified and labelled with Cy3 or Cy5 using a low RNA input Fluor Linear Amp Kit (Agilent Technologies, USA). Internal RNA controls from Agilent's RNA Spike-in Kit were included, comprised of mixtures of 11 in vitro synthesized transcripts derived from the Adenovirus E1A transcriptome at various ratios. The final cRNA concentration of 500 ng total RNA used was typically 590-650 ng/µl and the Cy3- or Cy5-cytidine incorporation was 5.4 -6.0 pmol/µg cRNA as determined using the Nanodrop spectrophotometer ND-1000. 3.5 µg of Cy3 and Cy5 labeled cRNA (treated vs. untreated samples) were competitively hybridized to high-density DNA arrays from Agilent Technologies (Whole Human Genome Oligo Microarray 44K) for 17 hours at 65°C according to the manufacturer's protocol. Each set of three microarray experiments for triplicate analysis contained one dye swap experiment. All microarrays were scanned and the intensities normalized over background as well as to eliminate signal intensity-dependent bias from the ratio of the two channels (Lowess normalization) using a microarray scanner from Agilent Technologies including proprietary software. All microarray data sets were imported into the microarray data analysis software Genespring 7.3 (Agilent Technologies) followed by comparison of normalized intensities. Gene identities were updated according to Agilent's latest list of gene annotations (G4112F, 07Feb07). Consistency of microarray data quality was assessed by monitoring performance of the spiked internal RNA controls (330 microarray spots/array) at various ratios (Supplementary Data, Fig. S1), by carrying out a global analysis of all absolute fluorescent intensity values, and by analysis of negative control spots (314 microarray spots/array) for determination of detection thresholds."
Comment[Study Grant Number]	""
Comment[Study Funding Agency]	""
Study Submission Date	""
Study Public Release Date	"2008-04-07"
Study File Name	"s_E-GEOD-10621_study_samples.txt"
STUDY DESIGN DESCRIPTORS
Study Design Type	"unknown_experiment_design_type"	"transcription profiling by array"
Study Design Type Term Accession Number	""	""
Study Design Type Term Source REF	""	""
STUDY PUBLICATIONS
Study PubMed ID	"20551148"
Study Publication DOI	"10.1152/physiolgenomics.00012.2010"
Study Publication Author List	"Reitmair A, Lambrecht NW, Yakubov I, Nieves A, Old D, Donde Y, Dinh D, Burk R, Sachs G, Im WB, Wheeler L."
Study Publication Title	"Prostaglandin E2 receptor subtype EP2- and EP4-regulated gene expression profiling in human ciliary smooth muscle cells."
Study Publication Status	"Published"
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STUDY FACTORS
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STUDY ASSAYS
Study Assay File Name	"a_E-GEOD-10621_GeneChip_assay.txt"
Study Assay Measurement Type	"transcription profiling"
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Study Assay Technology Type	"DNA microarray"
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STUDY PROTOCOLS
Study Protocol Name	"P-10621-3"	"P-10621-1"	"P-10621-2"	"P-10621-4"	"P-10621-5"	"P-10621-8"	"P-10621-6"	"P-10621-7"	"P-10621-9"
Study Protocol Type	"nucleic_acid_extraction"	"labeling"	"hybridization"	"feature_extraction"	"bioassay_data_transformation"	"bioassay_data_transformation"	"bioassay_data_transformation"	"bioassay_data_transformation"	"bioassay_data_transformation"
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Study Protocol Description	"Ambion RNAqueous RNA isolation kit"	"Low RNA Input Fluor Linear Amplification Kit (Agilent Technologies, USA)"	"Agilent 44K hybridization, 17 hrs"	"Agilent scanner and Feature Extraction Software"	"Data heading descriptions from GEO:<br>#ID_REF = <br>#VALUE = log2 of PRE_VALUE<br>#CH1_SIG = Control<br>#CH2_SIG = EP2 agonist<br>#RATIO = Ratio of EP2 agonist versus control"	"Data heading descriptions from GEO:<br>#ID_REF = <br>#VALUE = log2 of PRE_VALUE<br>#CH1_SIG = Cy3 (treated)<br>#CH2_SIG = Cy5 (control)<br>#RATIO = Ratio EP4 versus control"	"Data heading descriptions from GEO:<br>#ID_REF = <br>#VALUE = log2 of PRE_VALUE<br>#CH1_SIG = Cy3 (control)<br>#CH2_SIG = Cy5 (treated)<br>#RATIO = Ratio EP2 versus control"	"Data heading descriptions from GEO:<br>#ID_REF = <br>#VALUE = log2 of PRE_VALUE<br>#CH1_SIG = Cy3 (treated)<br>#CH2_SIG = Cy5 (control)<br>#RATIO = Ratio EP2 versus control (dye swap)"	"Data heading descriptions from GEO:<br>#ID_REF = <br>#VALUE = log2 of PRE_VALUE<br>#CH1_SIG = Cy3 (control)<br>#CH2_SIG = Cy5 (treated)<br>#RATIO = Ratio EP4 versus control (dye swap)"
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STUDY CONTACTS
Study Person Last Name	"Lambrecht"
Study Person First Name	"Nils Werner Georg"
Study Person Mid Initials	""
Study Person Email	"nilslam@ucla.edu; NLambrecht@mednet.ucla.edu"
Study Person Phone	""
Study Person Fax	""
Study Person Address	"Membrane Biology Laboratory, Physiology, UCLA, 11301 Wilshire Blvd, Bldg.113, Rm.325A, Los Angeles, 90073, USA"
Study Person Affiliation	"UCLA"
Study Person Roles	"submitter"
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Comment[Study Person REF]	""