Comment[ArrayExpressAccession]	E-GEOD-25927
Public Release Date	2010-12-14											
Investigation Title	Genome-wide analysis of alternative splicing in Caenorhabditis elegans											
Comment[Submitted Name]	Genome-wide analysis of alternative splicing in Caenorhabditis elegans
Experiment Description	Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provides the most comprehensive set of annotated splice variants in C. elegans to date, and is therefore expected to faciliate focused, high resolution in vivo functional assays of AS function. Alternative splicing events were identified from alignments of C. elegans mRNA/EST sequences (UniGene Build #26) to C. elegans genomic sequence (NCBI timestamp: Sept. 25, 2006), essentially as previously described (Pan et al. 2005; Pan et al. 2004). In total, 499 cassette type AS events were identified. For each AS event, 3 exon probes and 3 exon junction probes were designed to profile the AS event on the microarray, essentially as previously described (Pan et al. 2004).  This submission represents the expression microarray component of the study.											
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Term Source Name	EFO
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Term Source File	http://www.ebi.ac.uk/efo/efo.owl											
Person Last Name	Pan	Calarco	Ramani	Pan	Mavandadi	Wang	Nelson	Lee	Morris	Blencowe	Zhen	Fraser
Person First Name	Qun	John	Arun	Qun	Sepand	Ying	Andrew	Leo	Quaid	Benjamin	Mei	Andrew
Person Mid Initials		A	K				C	J		J		G
Person Email	qun.pan@utoronto.ca											
Person Affiliation	University of Toronto											
Person Phone	416-978-4633											
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Person Address	University of Toronto, , Toronto, Canada											
Person Roles	submitter											
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Protocol Name	P-GSE25927-1	P-GSE25927-5	P-GSE25927-4	P-GSE25927-2	P-GSE25927-3	P-GSE25927-6						
Protocol Description	ID_REF = <br>VALUE = Normalized asinh value	Slides were scanned using an Agilent Microarray Scanner (Agilent, Palo Alto, USA).	Mixed labeled cDNAs were added to hybridization buffer containing 1 M NaCl, 0.5% sodium sarcosine, 50 mM methyl ethane sulfonate (MES), pH 6.5, 33% formamide and 40 M-NM-<g herring sperm DNA (Invitrogen, Carlsbad, USA). Hybridizations were carried out in a final volume of 0.5 ml injecting into an Agilent hybridization chamber at 42M-0C on a rotating platform in a hybridization oven (Robbins Scientific Corporation, Sunnyvale, USA) for 16M-bM-^@M-^S24 h. Slides were then washed (rocking for approximately 30 seconds in 6 M-CM-^W SSPE, 0.005% sarcosine, then rocking for approximately 30 seconds in 0.06 M-CM-^W SSPE) and scanned with a 4000A microarray scanner (Axon Instruments, Union City, USA). Hybridizations were performed in duplicate or triplicate with fluor reversal: that is, each mRNA sample was examined 2-3 times, at least once in the Cy3 channel and at least once in the Cy5 channel, on separate arrays. Each array was hybridized with two samples simultaneously, each from a different developmental stage.	RNA was extracted using homogenization and Trizol reagent (Invitrogen, Carlsbad, USA) following the instructions from the manufacturer. Prior to cDNA synthesis, polyA+ RNA was purified from total RNA using the Nucleotrap mRNA Midi kit (Clontech) as recommended by the manufacturer with two rounds of purification.	The mRNA (1M-bM-^@M-^S2 M-NM-<g) was reverse-transcribed with random nonamer primers (1 M-NM-<g per reaction) and T18VN (0.25 M-NM-<g per reaction) to synthesize cDNA. The reaction contained a 1:1 mixture of 5-(3-aminoallyl) thymidine 5'-triphosphate (Sigma, St. Louis, USA) and thymidine triphosphate (TTP) in place of TTP alone. The cDNA products were bound to QIAquick PCR Purification columns (Qiagen, Hilden, Germany) following the manufacturer's instructions, washed three times with 80% ethanol, and eluted with water. Purified cDNA was reacted with N-hydroxy succinimide esters of Cy3 or Cy5 (Amersham Pharmacia Biotech, Piscataway, USA) following the manufacturer's instructions.	Images were processed by the Agilent Feature Extraction software (version 9.5). Variance stabilizing normalization (vsn) was performed using the vsn package within Bioconductor (version 2.1).						
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Protocol Type	bioassay_data_transformation	image_aquisition	hybridization	nucleic_acid_extraction	labeling	feature_extraction						
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Experimental Factor Name	DEVELOPMENTAL STAGE											
Experimental Factor Type	developmental stage											
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Publication Title	Genome-wide analysis of alternative splicing in Caenorhabditis elegans.											
Publication Author List	Ramani AK, Calarco JA, Pan Q, Mavandadi S, Wang Y, Nelson AC, Lee LJ, Morris Q, Blencowe BJ, Zhen M, Fraser AG											
PubMed ID	21177968											
Publication DOI	10.1101/gr.114645.110											
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Comment[SecondaryAccession]	GSE25927											
Comment[AdditionalFile:Data4]	GSE25927_US22502719_SLOT05_S02_GE2_95_robust_Leo_244k.txt											
Comment[AdditionalFile:Data1]	GSE25927_US22502719_SLOT01_S05_GE2_95_robust_Leo_244k.txt											
Comment[AdditionalFile:Data6]	GSE25927_US22502719_SLOT09_S01_GE2_95_robust_Leo_244k.txt											
Comment[GEOReleaseDate]	2010-12-14
Comment[AdditionalFile:Data5]	GSE25927_US22502719_SLOT08_S01_GE2_95_robust_Leo_244k.txt											
Comment[AdditionalFile:Data2]	GSE25927_US22502719_SLOT02_S03_GE2_95_robust_Leo_244k.txt											
Comment[AdditionalFile:Data3]	GSE25927_US22502719_SLOT05_S01_GE2_95_robust_Leo_244k.txt											
Comment[AdditionalFile:Data7]	GSE25927_readme.txt											
Comment[GEOLastUpdateDate]	2012-03-22
Comment[AEExperimentType]	transcription profiling by array											
Comment[ArrayExpressSubmissionDate]	2010-12-08
SDRF File	E-GEOD-25927.sdrf.txt