Comment[ArrayExpressAccession]	E-GEOD-53693
MAGE-TAB Version	1.1											
Public Release Date	2014-04-04
Investigation Title	Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation											
Comment[Submitted Name]	Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation
Experiment Description	Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptideM-bM-^@M-^Pencoding genes in vertebrates, providing an entry point to define their function in vivo. Ribosome profiling experiments at five timepoints across zebrafish development in WT embryos											
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	Giraldez	Bazzini	Johnstone	Christiano	Mackowiak	Obermayer	Fleming	Vejnar	Lee	Rajewsky	Walther	Giraldez
Person First Name	Antonio	Ariel	Timothy	Romain	Sebastian	Benedikt	Elizabeth	Charles	Miler	Nikolaus	Tobias	Antonio
Person Mid Initials	J.	A	G		D		S	E	T		C	J
Person Email	antonio.giraldez@yale.edu											
Person Affiliation	Yale University											
Person Phone	203 785 5423											
Person Address	Genetics, Yale University, 333 Cedar Street, New Haven, CT, USA											
Person Roles	submitter											
Protocol Name	P-GSE53693-3	P-GSE53693-2	P-GSE53693-1									
Protocol Description	Base calling was performed using CASAVA-1.8.2. The Illumina TruSeq adaptor sequence was then trimmed from raw reads by aligning its sequence, requiring 100% match of the first five base-pairs and a minimum alignment score of 60 (Matches:5, Mismatches:-4, Gap opening:-7, Gap extension:-7). Trimmed reads were then depleted of rRNA, tRNA, snRNA, snoRNA, and misc_RNA from Ensembl and RepeatMasker annotations using strand-specific alignment performed with Bowtie2 v2.1.0 with default parameters. Filtered reads were aligned strand-specific to the Zebrafish Zv9 genome assembly using Tophat v2.0.8 with default parameters and the exon-junction coordinates from Ensembl r70. Using the spliced version of each transcript (ensembl r73, Pauli et al lincRNAs, Ulitsky et al lincRNAs) we defined all ORFs as stop codon to most distal in-frame AUG codon without an intervening stop. Read coverage and counts were calculated using 28 & 29nt reads. For position-related calculation of ORFScore and Frame1 coverage, we counted reads only with the +12 position within the interior of the ORF (defined as the region excluding the reads aligning to the start and stop(-1) codons). RPKM values were calculated using input mRNA per transcript, normalizing WITHIN but not between transcript sets (total reads mapping to ensembl r73 or either lincRNA set). ORFScore and coverage values were calculated as outlined in Bazzini et al, 2013. Sequencing data were combined for each timepoint. Genome_build: Zv9/danRer7 Supplementary_files_format_and_content: allinfo_final_ORFs.txt :: orfID=unique ID of ORF; geneID; transcriptID; startLoc & stopLoc=1-based transcript coordinates of ORF; txCDSStart&txCDSEnd=start and end of annotated CDS; Source=transcript source; orfFrame=ORFScore; orfRate=# of reads in ORF; covF1Prop=proportion of in-frame positions with aligning read(s); tx_Input_RPKM=RPKM of transcript from input mRNA	For ribosome profiling, 50 wild type embryos for each condition were collected at a given stage. Embryos were lysed using 800ul of a mammalian cell lysis buffer containing 100ug/ml Cycloheximide as per the manufacturerM-bM-^@M-^Ys instruction (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For nuclease treatment, 3ul of ARTseq Nuclease was used. Ribosome protected fragments were run and 28-29nt fragments were gel purified as previously described in (Bazzini et al., 2012) and cloned according to the manufacturers protocol (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For RNA input, total RNA was isolated from 400 ul of the 800 ul of clarified extract before ART- seq Nuclease treatment. Poly-A selection was done according to manufacturer guide- lines (Dynabeads mRNA Purification Kit, Cat no.610.06), and RNA fragmented using the Artseq Ribosome Profiling Kit Mammalian protocol. Both RPF and RNA input fragments were cloned according to the Artseq Ribosome Profiling Kit, Mammalian. The final PCR was carried out with an initial 15 second denaturation at 98 M-0C, followed by 9-12 cycles of 15 seconds at 98 M-0C, 5 seconds at 55 M-0C and extension at 72 M-0C for 10 s. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel and DNA fragments of the correct size were extracted and sequenced.	Embryos are incubated at 28C									
Protocol Type	normalization data transformation protocol	nucleic acid library construction protocol	growth protocol									
Experimental Factor Name	STAGE	SEPARATION										
Experimental Factor Type	Stage	separation										
Publication Title	Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.											
Publication Author List	Bazzini AA, Johnstone TG, Christiano R, Mackowiak SD, Obermayer B, Fleming ES, Vejnar CE, Lee MT, Rajewsky N, Walther TC, Giraldez AJ											
PubMed ID	24705786											
Publication DOI	10.1002/embj.201488411											
Comment[SecondaryAccession]	GSE53693											
Comment[GEOReleaseDate]	2014-04-04											
Comment[ArrayExpressSubmissionDate]	2013-12-27											
Comment[GEOLastUpdateDate]	2014-04-11											
Comment[AEExperimentType]	RNA-seq of coding RNA											
Comment[AEExperimentType]	RNA-seq of non coding RNA											
Comment[AdditionalFile:Data1]	GSE53693_allinfo_final_ORFs.txt											
Comment[SecondaryAccession]	SRP034750											
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR1062197-SRR1062818											
SDRF File	E-GEOD-53693.sdrf.txt