Comment[ArrayExpressAccession]	E-GEOD-41900
MAGE-TAB Version	1.1			
Public Release Date	2013-08-08
Investigation Title	Phosphate-independent small RNA sequencing of WT and mir-249 mutant C. elegans			
Comment[Submitted Name]	Phosphate-independent small RNA sequencing of WT and mir-249 mutant C. elegans
Experiment Description	MicroRNAs (miRNA), discovered in C. elegans, are short non-coding RNAs that bind and regulate the expression of target mRNAs in animals and plants. C. elegans miRNAs bind to partially complementary sequences in the 3' UTR of the target mRNA, which results in translational repression through mRNA destabilization. The high-throughput sequencing of RNA cleavage fragments was performed to directly detect cleaved miRNA targets in C. elegans. Based on this analysis, miR-249 was identified as a potential miRNA that regulates a ZK637.6 pseudogene, paralogous to asna-1 (ZK637.5), by a cleavage mechanism with extensive, evolutionary conserved complementarity. Additionally, we validated miR-249-directed cleavage of the ZK637.6 by a gene-specific 5M-bM-^@M-^Y rapid amplification of cDNA ends and observed notable difference in expression of ZK637.6 in wild-type versus mir-249 mutant C. elegans. Furthermore, phosphate-independent small-RNA sequencing analysis revealed that miR-249 target genes, including ZK637.6 pseudogene, showed 22G-RNA productions dependent on miR-249 targeting. These findings may lead to a better understanding of the biological roles of miRNAs for pseudogenes in C. elegans. Total small RNAs from wild-type and mir-249 mutant in adult stage worms were subjected to small RNA sequencing using an Illumina platform with Tobacco Acid Pyrophosphatase (TAP) treatment, allowing detection of secondary siRNAs carrying 5M-bM-^@M-^Y-tri-phosphate.			
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	Shin	Park	Nam	Shin
Person First Name	Chanseok	June	Jin	Chanseok
Person Mid Initials		H	W	
Person Email	cshin@snu.ac.kr			
Person Affiliation	Seoul National University			
Person Address	Seoul National University, Gwanak-gu Daehak-dong, SEOUL, South Korea			
Person Roles	submitter			
Protocol Name	P-GSE41900-2	P-GSE41900-1		
Protocol Description	All small-RNA data were mapped to the genome and exon-junction regions, allowing no mismatches and at most 10 multiple mapping loci. If a read mapped to multiple loci, the read number was normalized to the number of loci. 9,298,952 and 9,232,069 reads from wild type adult and mir-249 mutant adult, respectively, were mapped to C. elegans genome (ce6). After excluding rRNAs, miRNAs, 21U RNAs, and other structural ncRNAs, the remaining small RNAs were classified into 22G RNAs and 26G RNAs based on their length and 5M-bM-^@M-2 terminal nucleotide. To identify targets, we used RefSeq gene annotations (ce6) updated with new 3M-bM-^@M-2 UTR annotations based on 3P-Seq data. Genome_build: C. elegans genome (ce6) Supplementary_files_format_and_content: The *bed files represent only the 22G-RNAs. The main purpose of employing the phosphate-independent small RNA sequencing was to analyze and compare the 22G-RNA populations in wild-type C. elegans versus mir-249 knockout mutant C. elegans. Abundance measurements are present in the *bed files (ie, in the 4th of 9 fields).	Worms were harvested at the appropriate stage, washed twice with M9 buffer, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 37M-0C for 10min, and total RNA was extracted using Tri-Reagent (Ambion). Isolated total RNA was subjected to DNAse treatment (Takara) and further purified with phenol:chloroform followed by precipitation with ethanol. Small RNA sequencing library construction was performed using ScriptMinerM-bM-^DM-" Small RNA-Seq Library Preparation Kit, according to manufacturer's instruction (Illumina). 5ug of total RNA from adult C. elegans was ligated to the 3' adaptor oligo, treated Tobacco Acid Pyrophosphatase (TAP), and subsequently ligated to the 5' adaptor oligo. Di-tagged RNA was then purified using Zymo RNA Clean & Concentrator Kits (Zymo Research). Reverse transcription was performed with MMLV Reverse Transcriptase for 15min at 37M-0C followed by heat inactivation for 15min at 85M-0C. The resulting cDNA was PCR-amplified for 18 cycles, with the ScriptMiner Multiplex Forward PCR primer and ScriptMiner Reverse PCR primer. The purified PCR product was submitted for sequencing.		
Protocol Type	normalization data transformation protocol	nucleic acid library construction protocol		
Experimental Factor Name	GENOTYPE			
Experimental Factor Type	genotype			
Publication Title	Degradome sequencing reveals an endogenous microRNA target in C. elegans.			
Publication Author List	Park JH, Ahn S, Kim S, Lee J, Nam JW, Shin C			
PubMed ID	23485825			
Publication DOI	10.1016/j.febslet.2013.02.029			
Comment[SecondaryAccession]	GSE41900			
Comment[GEOReleaseDate]	2013-08-08			
Comment[ArrayExpressSubmissionDate]	2012-10-29			
Comment[GEOLastUpdateDate]	2013-08-08			
Comment[AEExperimentType]	RNA-seq of non coding RNA			
Comment[SecondaryAccession]	SRP016865			
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR609440-SRR609441			
SDRF File	E-GEOD-41900.sdrf.txt