Comment[ArrayExpressAccession]	E-GEOD-40956
MAGE-TAB Version	1.1								
Public Release Date	2013-06-05
Investigation Title	Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [differentiation data]								
Comment[Submitted Name]	Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [differentiation data]
Experiment Description	This work provides the first evidence that Qk is a global regulator of splicing in vertebrates, defines a new splicing regulatory network in muscle, and suggests that overlapping splicing networks contribute to the complexity of changes in alternative splicing during differentiation. Alternative splicing contributes to muscle development and differentiation, but the complete set of muscle splicing factors and their combinatorial interactions are not known. Previously work identifies ACUAA (STAR motif) as an enriched sequence near muscle-specific alternative exons such as Capzb exon 9. We did mass spectrometry of proteins selected by wild type and mutant Capzb intron 9 RNA affinity chromatography, and identified Quaking (Qk), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We show that in myoblasts, Qk promotes inclusion of Capzb exon 9 in opposition to repression by PTB. Qk knockdown in myoblasts has little effect on transcript levels, but alters inclusion of 824 cassette exons whose adjacent intron sequences are enriched in ACUAA motifs. During differentiation to myotubes, Qk levels increase 2-3 fold, suggesting a mechanism for Qk-responsive exon regulation.  We captured the PTB splicing regulatory network and intersected it with the Qk network, identifying overlap between the functions of Qk and PTB. Approximately 60% of exons whose inclusion is altered during myogenesis appear to be under control of one or both of these splicing factors in myoblasts. This series is the C2C12 differentiation data. It is 9 arrays, 3 timepoints, with 3 replicates. The time points are 0 hrs, 24 hrs, and 72hrs.								
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl							
Person Last Name	Ares	Ares Jr	Hall	Nagel	Fagg	Shiue	Cline	Perriman	Donohue
Person First Name	Manny	Manny	Megan	Roland	Samuel	Lily	Melissa	Rhonda	John
Person Mid Initials							S	J	P
Person Email	geo@ncbi.nlm.nih.gov								
Person Affiliation	UCSC								
Person Address	Molecular and Cellular Biology, UCSC, 1125 High St, Santa Cruz, CA, USA								
Person Roles	submitter								
Protocol Name	P-GSE40956-1	P-GSE40956-5	P-GSE40956-6	P-GSE40956-2	P-GSE40956-3	P-GSE40956-4	P-GSE40956-7		
Protocol Description	Intensity was calculated by AffyPowerTools using dabg , gc background correction, iter-plier and quantile normalization Data was analyzed according to the methods in Sugnet et al. PLoS Comput Biol. 2006 Jan;2(1):e4. PMID: 16424921 probe group file: mjay.clf meta-probeset file: mjay.pgf ID_REF =  VALUE = Intensity as calculated by AffyPowerTools	Amplified, labeled cDNA was produced using the GeneChipM-. Whole Transcript Sense Target Labeling and Control Reagents (Affymetrix) according to the manufacturerM-bM-^@M-^Ys instructions	The labeled, amplified cDNA was hybridized overnight to the MJAY Chip (Affymetrix)	Differentiation was verified visually by myotube formation and via Western blot to visualize myogenic markers. RNA and protein were isolated either immediately prior to (zero time) or up to 72 hours after addition of differentiation medium.	Mouse C2C12 myoblast cells (ATCC) were maintained in DulbeccoM-bM-^@M-^Ys modified EagleM-bM-^@M-^Ys medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen). To induce myogenesis, 1.5 x 105 cells were plated in growth medium (DMEM + 10% FBS) per well in 6-well tissue culture-treated plates (Fisher) 12-18 hours prior to the addition of differentiation medium. Cells were washed once with differentiation medium (DMEM supplemented with 2% horse serum [Invitrogen]) prior to maintenance in the same medium. Media was changed every 24 hours, and myotube formation was visible	Ribosomal RNAs were removed from three replicate individual total RNA preparation for each sample using the RiboMinus Transcriptome Isolation Kit (Invitrogen) according to the manufacturers instructions.	Chips were washed and labeled using the Affymetrix Fluidics Station 450, and scanned on an Affymetrix GeneChip scanner.		
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol		
Experimental Factor Name	TIME DIFFERENTIATED								
Experimental Factor Type	time differentiated								
Publication Title	Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation.								
Publication Author List	Hall MP, Nagel RJ, Fagg WS, Shiue L, Cline MS, Perriman RJ, Donohue JP, Ares M Jr								
PubMed ID	23525800								
Publication DOI	10.1261/rna.038422.113								
Comment[SecondaryAccession]	GSE40956								
Comment[GEOReleaseDate]	2013-06-05								
Comment[ArrayExpressSubmissionDate]	2012-09-18								
Comment[GEOLastUpdateDate]	2013-06-05								
Comment[AEExperimentType]	transcription profiling by array								
SDRF File	E-GEOD-40956.sdrf.txt