Comment[ArrayExpressAccession]	E-GEOD-56180
MAGE-TAB Version	1.1								
Public Release Date	2014-05-23
Investigation Title	Unambiguous Identification of miRNA:target site Interactions by Different Types of Ligation Reactions								
Comment[Submitted Name]	Unambiguous Identification of miRNA:target site Interactions by Different Types of Ligation Reactions
Experiment Description	To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (“re state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C. elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in absence of the exogenous ligase. Our in vivo dataset and re-analysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded >17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, non-canonical, and non-conserved miRNA interactions. Our data suggest that ~80% of miRNA:targets have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, in vivo discovery of miRNA interactions. In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011) using GFP-tagged ALG-1 expressing worms in L3 stage. Worm lysate was treated with RNase T1. Following immunoprecipitation and a second RNase T1 digest, it was proceeded as described in Hafner et al. 2010. For the modified iPAR-CLIP ligation samples and its control samples immuno-purified complexes were treated with PNK phospathase minus, subjected to ligation with T4 RNA ligase/no ligase added and subsequently phosphorylated with PNK. Protein purification and RNA library preparation essentially as described in Hafner et al., but with the selection of longer RNA products.								
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	Rajewsky	Grosswendt	Filipchyk	Manzano	Klironomos	Schilling	Herzog	Gottwein	Rajewsky
Person First Name	Nikolaus	Stefanie	Andrei	Mark	Filippos	Marcel	Margareta	Eva	Nikolaus
Person Email	rajewsky@mdc-berlin.de								
Person Affiliation	Max Delbrück Center for Molecular Medicine								
Person Phone	+49 30 9406-2998								
Person Address	Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, Berlin, Berlin, Germany								
Person Roles	submitter								
Protocol Name	P-GSE56180-2	P-GSE56180-1							
Protocol Description	removal of 3'adapter sequence with FLEXBAR (see supplementary methods) collapsing distinct read-sequences with custom script searching anchors of the left part of reads in mirbase ce6 mature sequences with custom script alignment of the right part of reads with BOWTIE2 2.1.0 to ce6 AGL-1 PAR-CLIP clusters miRNA:targets identification and quality filtering to limit false-discovery rate with custom scripts Genome_build: ce6 Supplementary_files_format_and_content: GFF file with genomic regions of target parts of miRNA:targets	in vivo PAR-CLIP (Jungkamp et al. 2011) Hafner et al. 2010							
Protocol Type	normalization data transformation protocol	nucleic acid library construction protocol							
Experimental Factor Name	3' ADAPTER								
Experimental Factor Type	3' adapter								
Comment[SecondaryAccession]	GSE56180								
Comment[GEOReleaseDate]	2014-05-23								
Comment[ArrayExpressSubmissionDate]	2014-03-25								
Comment[GEOLastUpdateDate]	2014-05-23								
Comment[AEExperimentType]	RNA-seq of non coding RNA								
Comment[AdditionalFile:Data1]	GSE56180_interactions_all-samples.gff								
Comment[SecondaryAccession]	SRP040587								
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR1207389-SRR1207395								
SDRF File	E-GEOD-56180.sdrf.txt