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| Status |
Public on Mar 31, 2016 |
| Title |
heart |
| Sample type |
SRA |
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| Source name |
adult heart (1 year old)
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| Organism |
Danio rerio |
| Characteristics |
strain/background: AB
genotype/variation: wild type
tissue: heart
developmental stage: adult
age: 1 year
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| Growth protocol |
Wild type AB zebrafish strain was maintained at 28ºC on a 14 h-light/10 h-dark cycle.
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| Extracted molecule |
total RNA |
| Extraction protocol |
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 3' adapter (AMP-5'p-5'p/CTGTAGGCACCATCAATdi-deoxyC- 3') in absence of ATP and gel excised in the range of 35 and 50 nt. A second ligation was performed with the 5' adapter ("Nelson's linker" 5'ATCGTrArGrGrCrArCrCrUrGrArArA 3'), for 1 hour at 37°C, followed by phenol extraction. First strand cDNA synthesis was then performed using a specific 3'-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.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
454 GS FLX |
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| Data processing |
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 3' 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 after data processing for each sample.
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| Submission date |
Mar 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ana Soares |
| E-mail(s) |
ana.r.soares@ua.pt
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| Organization name |
University of Aveiro
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| Street address |
Campus de Santiago
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| City |
Aveiro |
| ZIP/Postal code |
3810 |
| Country |
Portugal |
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| Platform ID |
GPL19872 |
| Series (1) |
| GSE66718 |
Identification of small non-coding RNAs in zebrafish |
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| Relations |
| BioSample |
SAMN03396655 |
| SRA |
SRX915249 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1630513_heart_processed.txt.gz |
1007 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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