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| Status |
Public on Jul 29, 2014 |
| Title |
JM15_1 |
| Sample type |
SRA |
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| Source name |
total RNA extracted from brain
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| Organism |
Nothobranchius furzeri |
| Characteristics |
age: 39 weeks
tissue: brain
strain: MZM 04/10
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| Extracted molecule |
total RNA |
| Extraction protocol |
The whole fish brains were dissected and transferred into 2 ml tubes with 1 ml cooled QIAzol (Qiagen, Hilden, Germany) and one 5 mm stainless steel bead (Qiagen) was added. Homogenization was performed using a TissueLyzer II (Qiagen) at 20 Hz for 2-3x 1 min. After incubation for 5 min at room temperature 200 µl chloroform was added. The tube was shaken for (at least) 15 s and incubated for 3 min at room temperature. Phase separation was achieved by centrifugation at 12,000x g for 20 min at 4°C. The aqueous phase was transferred into a fresh cup and 10 µg of Glycogen (Invitrogen, Darmstadt, Germany), 0.16x volume NaAc (2 M; pH 4.0) and 1.1x volume isopropanol were added, mixed thoroughly and incubated for 10 min at room temperature. The RNA was precipitated by a centrifugation step with 12,000 x g at 4°C for 20 min. The supernatant was removed and the pellet was washed with 80% Ethanol twice and air dried for 10 min. The RNA was resuspended in 20 µl DEPC-treated water by pipetting up and down, followed by incubation at 65°C for 5 min. The RNA was quantified with a NanoDrop 1000 (PeqLab, Erlangen, Germany) and stored at -80°C until use.
Sequencing procedure was done using Illumina methodology [Bentley et al, 2008]. Around 2.5 µg of total RNA was used for library preparation (Illumina, TruSeq™ RNA Sample Prep Kit v2) using the manufacturer’s description.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - Illumina Casava software used for extraction of FASTQ files
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - FASTQ files were mapped using Bowtie versus a transcriptome catalogue of N. furzeri containing 19,812 transcripts
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - Uniquely mapped reads for each transcript were counted using R Statistical Language and Bioconductor.
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - The read counts were normalized with respect to the size of the individual genes and to the total amount of mappable reads obtained in the sequencing process, resulting in RPKM-values (Mortazavi et al. 2008) for each gene.
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - The transcripts were annotated additionally using Ensembl gene identifiers from D. rerio, while 13,561 shared a best bi-directional BLAST hit between both species.
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - Genome_build: Transcriptome sequence published by Petzold et al. 2013 [PMID: 23496936]
GSE52462_Nfurzeri_MZM_brain_Normal_Ageing_counts_RPKM.xls - Supplementary_files_format_and_content: Excel file including raw counts and RPKM values for each sample
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GSE52462_counts_RPKMs_2015.xls - Illumina Casava software used for extraction of FASTQ files
GSE52462_counts_RPKMs_2015.xls - FASTQ files were mapped using Tophat (v2.0.6) to the N.furzeri genome.
GSE52462_counts_RPKMs_2015.xls - Uniquely mapped reads were counted for each gene_id using featureCounts v1.4.3-p1 (featureCounts -T 8 -a $GTF -s 0 -t exon -g gene_id -o featureCounts_gene.txt $SAMFILE)
GSE52462_counts_RPKMs_2015.xls - RPKM values were computed using exon lengths provided by featureCounts as described in Mortazavi et al. 2008.
GSE52462_counts_RPKMs_2015.xls - Genome_build: genome v20150522 and annotation v20140422 can be downloaded from the Nothobranchius furzeri Information Network Genome Browser (http://nfingb.fli-leibniz.de/)
GSE52462_counts_RPKMs_2015.xls - Supplementary_files_format_and_content: Excel file includes raw counts and RPKM values
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| Submission date |
Nov 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
JenAge Project |
| E-mail(s) |
[email protected]
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| Organization name |
Leibniz Institute for Age Research - Fritz Lipmann Institute
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| Street address |
Beutenbergstr. 11
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| City |
Jena |
| ZIP/Postal code |
D-07745 |
| Country |
Germany |
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| Platform ID |
GPL17944 |
| Series (1) |
| GSE52462 |
RNA-seq of the brain of Nothobranchius furzeri at different time points |
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| Relations |
| BioSample |
SAMN02404744 |
| SRA |
SRX378355 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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