Comment[ArrayExpressAccession] E-GEOD-46512 MAGE-TAB Version 1.1 Public Release Date 2013-05-23 Investigation Title Ribosome Profiling over a Zebrafish Developmental Timecourse Comment[Submitted Name] Ribosome Profiling over a Zebrafish Developmental Timecourse Experiment Description To experimentally-validate the non-coding status of annotated lncRNAs, we performed ribosome profiling over a developmental timecourse that matched our previously-published (Pauli et al. 2012) developmental transcriptome. We find that many previously-annotated lncRNAs appear to be translated, but in a pattern more akin to 5' leaders of coding genes. Ribosome profiling over 8 stages in early zebrafish development: 2-4 cell, 256 cell, 1K cell, Dome, Shield, Bud, 28hpf and 5dpf 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 Chew Chew Pauli Valen Schier Person First Name Guo-Liang Guo-Liang Andrea Eivind Alexander Person Email chew@fas.harvard.edu Person Affiliation Harvard University Person Address Dept of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave BL 1019, Cambridge, MA, USA Person Roles submitter Protocol Name P-GSE46512-4 P-GSE46512-1 P-GSE46512-3 P-GSE46512-2 Protocol Description Adapters trimmed using custom script Reads that mapped to zebrafish rRNAs (SILVA rRNA database: http://www.arb-silva.de/) by Bowtie2 -N 1 -L 20 _k 20) were discarded Remaining reads mapped to previously assembled zebrafish developmental transcriptome (Pauli et al. 2012) on top of the Zv9/danRer7 assembly of the zebrafish genome by Tophat2; no indels, no novel junctions, -M -g 10 Only RPFs of length 27nt to 32nt used. P-site positions determined to be +12 for 27-28nt RPFs, +13 for 29-31nt RPFs, +13 for 29-31nt RPFs, +14 for 32nt RPFs. Custom scripts used to generate bw files of genome-wide ribosome profiles corresponding to P-site occupancy, in conjunction with BedTools and UCSC bedgraphToBedBed Genome_build: Zv9 (danRer7) Supplementary_files_format_and_content: bigwig files corresponding to P-site occupancy by ribosomes Embryos were quickly washed in ice cold PBS and flash frozen in liquid nitrogen Embryos were lysed by repeated micropipetting in 1.5ml of cold polysome buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1mM dithiothreitol, 100 μg/ml cycloheximide) with added 0.5% Triton X-100, 500 μg/ml GMP-PNP, 24 U/ml TurboDNase (Ambion AM2238), incubated with agitation for 10 min at 4°C, and clarified by centrifugation at 1300 rcf for 10 min at 4°C. 20μl RNAseI (Ambion AM2294) was added to the 1.5 ml of supernatant and incubated for 30 min at 37°C, then stopped by chilling on ice and addition of 40 μl of SuperaseIn (Ambion AM2694). Footprinted samples were pelleted through a sucrose cushion (1M sucrose in polysome buffer with added 100 U/ml SuperaseIn) by centrifugation at 260,000 rcf for 4.5 hours at 4°C, and resuspended in 800 μl 10mM Tris pH 7.4 with 1% SDS. RNA was purified by hot acid phenol/chloroform extraction and precipitated by standard ethanol precipitation. Libraries were prepared essentially as described by Ingolia et al. Cell 2011 400-600 embryos per stage from TL/AB WT strains were allowed to grow at 28.5°C and staged according to Kimmel et al. Dev. Dyn. 1995 Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name DEVELOPMENTAL STAGE Experimental Factor Type developmental stage Comment[SecondaryAccession] GSE46512 Comment[GEOReleaseDate] 2013-05-23 Comment[ArrayExpressSubmissionDate] 2013-04-30 Comment[GEOLastUpdateDate] 2013-05-26 Comment[AEExperimentType] RNA-seq of non coding RNA Comment[AdditionalFile:Data1] GSE46512_File_descriptions.txt Comment[AdditionalFile:Data2] GSE46512_mm9_lncLoci.gtf Comment[AdditionalFile:Data3] GSE46512_zv9_lncLoci.gtf Comment[SecondaryAccession] SRP021915 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR836192-SRR836199 SDRF File E-GEOD-46512.sdrf.txt