{"database": "metadata", "table": "run_metadata", "rows": [[36300, "SRR392999", "SRX113356", "SRS284300", "SRP010040", "PRJNA150397", "Ribosome profiling of early zebrafish embryos    miRNA mediated regulation during embryogenesis causes translational repression before mRNA decay", "GSE34743", "Transcriptome Analysis", "MicroRNAs regulate gene expression through deadenylation  repression and mRNA decay. However  the contribution of each mechanism in non steady state situations remains unclear.  We monitored the impact of miR 430 on ribosome occupancy of endogenous mRNAs in wild type and dicer mutants lacking mature miR 430. Our results indicate that miR 430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation  and disrupting deadenylation using an internal polyA tail did not block target repression. Finally  we observe that ribosome density along the length of the target mRNA remains constant  suggesting that translational repression occurs by reducing the initiation rate rather than reducing elongation or causing ribosomal drop off. In summary  our results show that miR 430 regulates translation initiation before inducing mRNA decay. Overall design: Time course parallel ribosome profiling and input mRNA quantification in wildtype and MZdicer mutant embryos", null, "pubmed:22422859", null, "Ribosome protected fragments Wild type 2hpf", "GSM854438", null, "source name:Whole embryo  wild type|sample type:ribosome protected fragments|tissue:whole embryo|genotype:wild type|development stage:2hpf", "Ribosome protected fragments Wild type 2hpf", "Ribo Wt 2 A.bed; genome build: danRer7 Tophat v1.2.0 alignment to UCSC danRer7 Zv9 genome sequence  guided by Ensembl r63 gene models; default settings  limited to reads mapping 5 or fewer times. Reads were trimmed of three prime adapter sequence", "Whole embryo  wild type", null, "Ribosome protected fragments were obtained from 80 embryos per sample according to an adapted protocol from Ignolia et al  2009. Embryos were incubated with 0.1 ug/ml cycloheximide in Methylene blue water for 5 min  then snap frozen in 25ul Lysis buffer and clarified for 10 min at 10g 4degC. Supernatant was incubated with RNaseI and DNase at room temperature for 45 minutes with gentle mixing  then digested extracts 50ul were overlain in a 100ul cushion of 1.8M sucrose in 20mM total Tris HCL ph8.0; 140mM KCl; 5 mM MgCl2; 100 ug/ml Cycloheximide; 0.5 mM DTT; 40U/ml SUPERas In Ambion #AM2694 for 14 h at 55.000 rpm in a TLS55 rotor in a Beckman TL100 ultracentrifuge. The bottom 30 ul was mixed with 4ul of PNK buffer and 4 ul of Proteinase K and incubated at 37degC for 30 minutes. RNA was extracted using Trizol  then run on a 15% Urea gel to excise the 28 38 nt region. To construct barcoded Illumina small RNA libraries  samples were eluted overnight in 300 mM NaOAc pH 5.5; 1 mM EDTA; 0.1U/ul SUPERas In Ambion #AM2694  followed by Ethanol precipitation. RFP and RNA input fragments were 3? dephosphorylated with polynucleotide kinase in T4 polynucleotide kinase buffer without xxx for 1 h at 37degC  followed by 10 min incubation at 75degC and ethanol precipitation. Samples were resuspended and ligated to Illumina v1.5 three prime adapter using T4 RNA Ligase 2  truncated K227Q  and RNAseOUT Recombinant Ribonuclease Inhibitor for 6h at 20degC  then separated on a 10% Urea gel to extract the three prime ligation products. These were five prime phosphorylated with polynucleotide kinase in T4 polynucleotide kinase buffer with 1mM ATP for 30 min at 37degC and precipitated in ethanol. five prime ligation was performed using T4 RNA Ligase 1 for 6 hrs at 20degC with individual 100uM barcoded five prime adapters  then separated on a 10% Urea gel. Gel purified samples were reverse transcribed using SuperScript II Reverse Transcriptase according to the Invitrogen protocol  then amplified using Phusion High Fidelity DNA Polymerase. PCR was carried out with an initial 30s denaturation at 98degC followed by 4 cycles of 30s denaturation at 98degC and 15s extension at 72degC  followed by 10 15 cycles of 10s denaturation at 98degC  30s annealing at 50degC  and 15s extension at 72degC. Reactions were separated on a non denaturing 8% polyacrylamide TBE gel and correct sized DNA fragments were sequenced using Illumina HiSeq2000. In parallel  RNA input from 20 embryos was extracted using Trizol  then polyA selected using biotinylated oligodT40 at room temperature and eluted. Samples were then alkaline fragmented for 20min at 95degC using 2 mM EDTA; 10 mM Na2CO3; 90 mM NaHCO3 pH 9.3; then neutralized with 560ul of ice cold NaOAc 300mM pH 5.5 and RNA precipitated with isopropanol. Small RNA sequencing libraries were constructed as above  selecting for fragments in the 30 50 nt range.", null, "sample type:ribosome protected fragments|tissue:whole embryo|genotype:wild type|developmental stage:2hpf", "GSM854438", "GSM854438: Ribosome protected fragments Wild type 2hpf; Danio rerio; RNA Seq", "GSM854438 1", "GSM854438: Ribosome protected fragments Wild type 2hpf", "1", null, "GEO Accession:GSM854438", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina Genome Analyzer II", "<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>72</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>", "SRP010040", null, "instrument model:Illumina HiSeq 2000", "Ribo_Wt_2_A2.fq", "fastq", 1197987552.0, 16638716.0, "GSM854438 r2", "0:72", "A:346631182;C:288138913;G:296030268;T:267150788;N:36401", 72, null, null, null, 346631182, 288138913, 296030268, 267150788, 36401, "SRX113356", "SRS284300", "SRA048903", "GEO", "Giraldez Lab, Genetics, Yale University", 1, 3e-05, null, 0.0, null, 0.99997, null, 0.0, null, 72, null, "T", null, "under 1.2% mapping rate", "illumina", "early_illumina", "unknown", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2011-12-27", "Cleavage", "Embryo", "Whole Organism", "All anatomical structures"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["36300"], "units": {}, "query_ms": 8.219092000217643}