run_metadata: 39617

This data as json

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
39617	SRR1873563	SRX915243	SRS870231	SRP056014	PRJNA277780	Identification of small non coding RNAs in zebrafish	GSE66718	Transcriptome Analysis	MicroRNAs miRNAs are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes namely developmental timing cell differentiation cell proliferation immune response and infection. For this reason their identification is essential to understand eukaryotic biology. Their small size low abundance and high instability complicated early identification; however cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNA identification in many eukaryotes. We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish Danio rerio. For this a series of cDNA libraries were prepared from small non coding RNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics and 25 novel miRNAs were predicted. Overall design: Small RNA libraries were prepared from different zebrafish developmental stages namely 24 hpf 72 hpf 96 hpf 5 dpf dpf 45 dpf young adult and from adult brain eyes gills heart skin and fins.		pubmed:26694924		5dpf	GSM1630507		source name:5 dpf embryos\|strain/background:AB\|genotype/variation:wild type\|tissue:whole body\|developmental stage:embryo\|age:5dpf	5dpf	Base calling and quality trimming of sequence reads was carried out using the Genome Sequencer FLX software. Raw images were processed to remove background noise and the data was normalized. TAGs and adapter sequences of zebrafish developmental and adult tissues samples were then identified and trimmed and those reads with correct TAGs and adapters > 15 nt were retrieved for downstream analysis using the miRDeep software https://www.mdc berlin.de/8551903/en/. miRDeep aligned the sequences against the zebrafish genome using megaBlast with seed length set at 12 the traditional blast output and minimum local identity set at 100. The blast output was then parsed for miRDeep uploading and aligned sequences with a maximum of 2 mismatches in the three prime end were retrieved. Reads that matched more than 10 different genome loci were discarded and only those with one or more alignments were kept and using the remaining alignments as guidelines the potential precursors were excised from the genome. The secondary structure of putative precursors was predicted using RNAfold and signatures were created by retaining reads that aligned perfectly with those putative precursors to generate the signature format. Finally miRDeep predicted miRNAs by discarding non plausible Dicer products and scoring plausible ones. To assess seed conservation plausible Dicer processing sequences were blasted against a local version of mature miRNAs from miRBase 12.0 that lacked zebrafish miRNA sequences. Borderline miRNA candidates were also resolved by determining their relative stability using Randfold. To distinguish between novel and known miRNAs selected pre miRNAs were blasted against Danio rerio stem loop sequences miRBase and those that did not produce any or produced imperfect alignments were scored as novel miRNAs. Pairs of signatures and structures were used to estimate the number of false positives by randomly permutation using miRDeep. To overcome the inherent lack of sensitivity of miRDeep novel transcripts encoding miRNAs predicted by bioinformatics were retrieved from Ensembl 5.2 using BioMart and from literature predictions. These sequences were then used to perform a megaBlast search against our data with seed length set at 12. The transcripts with perfect matches and alignment length larger than 18 nt were kept for further processing. These transcripts were then compared with the mature miRNAs present in miRBase 12.0 and those that produced imperfect alignments or did not produce alignments were considered new miRNAs. Read numbers were normalized as described by Chen and colleagues Genes & Development 2005 19:1288 1293 and a miRNA expression profile using identical number of reads for each sample was generated. The number of reads between samples was normalized as indicated below: Expression Reads = [1000 x NRmiRNAXY]/ TNRmiRNAsY where NRmiRNAXY is the number of reads of miRNAX X = any miRNA in sample Y and TNRmiRNAsY is the total number of miRNAs in sample Y. 1000 is an arbitrary number of reads. The data was transformed into log2 scale to build the heat map using the MeV 4.0 software package http://www.tm4.org/mev.html. Genome build: Zv8 Supplementary files format and content: Tab delimited text files include the miRNA IDs sequences and respective raw counts post data processing for each sample.	5 dpf embryos		100 μg of total RNA from each sample was isolated using TRIzol® and small RNAs were enriched by differential precipitation using polyethylene glycol. Total RNAs were fractioned using 12% denaturing PAGE and small RNAs of 15 30 nt were gel isolated using Gel Filtration cartridges from Edge Biosystems. For cDNA synthesis the small RNA molecules previously isolated were first ligated to a three prime adapter AMP five primep five primep/CTGTAGGCACCATCAATdi deoxyC three prime in absence of ATP and gel excised in the range of 35 and 50 nt. A second ligation was performed with the five prime adapter "Nelson's linker" five primeATCGTrArGrGrCrArCrCrUrGrArArA three prime for 1 hour at 37°C followed by phenol extraction. First strand cDNA synthesis was then performed using a specific three prime primer and Superscript™ III reverse transcriptase Invitrogen. RNase H treated cDNA was PCR amplified with adapter specific primers. Each sample contained a specific TAG constituted by 3 nucleotides as detailed next. 24hpf ATC; 72hpf ACT; 96hpf CAG; 5dpf ATG; 45dpf CCG; entire adult GTA; brain CGG; Heart CTG; Eyes GCT; fins GTT; skin TAC; gills TCC. PCR products were then run on 10% denaturing PAGE containing 7 M urea and the corresponding band 100 nt was eluted from the gel with Probe Elution Buffer from Ambion at 37°C overnight. These products were used for the emulsion PCR. Parallel DNA pyrosequencing was performed using the Genome Sequencer FLX Roche following established protocols for DNA library sequencing.	Wild type AB zebrafish strain was maintained at 28ºC on a 14 h light/10 h dark cycle.	strain/background:AB\|genotype/variation:wild type\|tissue:whole body\|developmental stage:embryo\|age:5dpf	GSM1630507	GSM1630507: 5dpf; Danio rerio; miRNA Seq	GSM1630507		1	100 μg of total RNA from each sample was isolated using TRIzol® and small RNAs were enriched by differential precipitation using polyethylene glycol. Total RNAs were fractioned using 12% denaturing PAGE and small RNAs of 15 30 nt were gel isolated using Gel Filtration cartridges from Edge Biosystems. For cDNA synthesis the small RNA molecules previously isolated were first ligated to a three prime adapter AMP five primep five primep/CTGTAGGCACCATCAATdi deoxyC three prime in absence of ATP and gel excised in the range of 35 and 50 nt. A second ligation was performed with the five prime adapter "Nelson's linker" five primeATCGTrArGrGrCrArCrCrUrGrArArA three prime for 1 hour at 37°C followed by phenol extraction. First strand cDNA synthesis was then performed using a specific three prime primer and Superscript™ III reverse transcriptase Invitrogen. RNase H treated cDNA was PCR amplified with adapter specific primers. Each sample contained a specific TAG constituted by 3 nucleotides as detailed next. 24hpf ATC; 72hpf ACT; 96hpf CAG; 5dpf ATG; 45dpf CCG; entire adult GTA; brain CGG; Heart CTG; Eyes GCT; fins GTT; skin TAC; gills TCC. PCR products were then run on 10% denaturing PAGE containing 7 M urea and the corresponding band 100 nt was eluted from the gel with Probe Elution Buffer from Ambion at 37°C overnight. These products were used for the emulsion PCR. Parallel DNA pyrosequencing was performed using the Genome Sequencer FLX Roche following established protocols for DNA library sequencing.	GEO Accession:GSM1630507	miRNA-Seq	TRANSCRIPTOMIC	size fractionation	SINGLE	LS454	454 GS FLX		SRP056014					168981.0	3060.0	GSM1630507 r1	0:4 1:51.22	A:51895;C:42870;G:40864;T:32741;N:611	4	51			51895	42870	40864	32741	611	SRX915243	SRS870231	SRA246117	GEO	University of Aveiro	1	0.0		0.0		1.0				50		T		under 1.2% mapping rate	legacy	early	3prime	size_fractionation	unknown	bulk	other_seq	454		Portugal	2015-03-09	Larval	Larval	Trunk	Surface Structure