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Sample GSM1693136

Query DataSets for GSM1693136

Status

Public on Dec 03, 2015

Title

JG13_5

Sample type

SRA

Source name

skin

Organism

Nothobranchius furzeri

Characteristics

age: 12 weeks
tissue: skin
strain: GRZ

Extracted molecule

total RNA

Extraction protocol

The organs of the fishes 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 30 Hz for 3x 1 min. After incubation for 5 min at room temperature 200 µl chloroform was added. The tube was shaken for 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

*******************
File: GSE69122_Nfurzeri_GRZ_BrainLiverSkin_NormalAgeing_12weeks_counts_RPKM.xls
Illumina Casava v1.8.2 software used for extraction of FASTQ files
FASTQ files were mapped using Bowtie versus a transcriptome catalogue of N. furzeri containing 19,812 transcripts
Uniquely mapped reads for each transcript were counted using R Statistical Language and Bioconductor.
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.
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.
genome build: Transcriptome sequence published by Petzold et al. 2013 [PMID: 23496936]
processed data files format and content: Excel file including raw counts and RPKM values for each sample
*******************
File: GSE69122_count_RPKMs_genome.xls
Illumina Casava v1.8.2 software used for extraction of FASTQ files
FASTQ files were mapped using Tophat (v2.0.6) to the N.furzeri genome.
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)
RPKM values were computed using exon lengths provided by featureCounts as described in Mortazavi et al. 2008.
genome build: genome v20150522 and annotation v20140422 can be downloaded from the Nothobranchius furzeri Information Network Genome Browser (http://nfingb.fli-leibniz.de/)
processed data files format and content: Excel file includes raw counts and RPKM values
*******************

Submission date

May 21, 2015

Last update date

May 15, 2019

Contact name

JenAge Project

E-mail(s)

[email protected]

Organization name

Leibniz Institute for Age Research - Fritz Lipmann Institute

Street address

Beutenbergstr. 11