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| Status |
Public on Jan 19, 2014 |
| Title |
xtr kd M1 [batch1] |
| Sample type |
SRA |
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| Source name |
Kidney
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| Organism |
Xenopus tropicalis |
| Characteristics |
Sex: male
strain: NA
age: adult (4-6 months)
extract_protocol: Illumina GAIIx, strand-specific
library-type (for tophat and cufflinks): fr-secondstrand
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using the Trizol (Invitrogen) procedure or RNAeasy/RNAeasy Lipid/miRNeasy (Qiagen) column purification kits. RNA quality was assessed using an Agilent 2100 Bioanalyzer.
Non-strand-specific sequencing libraries were prepared using the mRNA-Seq Sample Prep Kit (Illumina) according to the manufacturer's instructions. Briefly, polyadenylated RNA was isolated using a poly-dT bead procedure and then chemically fragmented and randomly primed for reverse transcription. After second-strand synthesis, the ends of the double-stranded cDNA were repaired. After 3'-end adenylation of these products, Illumina Paired-End Sequencing adapters were ligated to the blunt ends of the cDNA fragments. Ligated products were run on gels; 250-300 bp fragments were excised and then PCR-amplified (15 cycles). After column-purification, qualities of the resulting libraries were assessed using Agilent 2100 Bioanalyzers. Strand-specific libraries were prepared using an optimized version of the original Directional mRNA-seq Library Prep Pre-Release Protocol from Illumina. We used reagents from various companies instead of Illumina’s to decrease significantly the library cost and as a consequence adapted the reaction parameters for each step. In addition, we integrated a size selection step, right after fragmentation to optimize the insert size distribution and decrease the adapter contamination. This optimized protocol is available upon request.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
The basecalling was performed with the Illumina GAIIx pipeline, version 1.8 and with Ibis (Kircher et al, 2009). Ibis was used on non-strand specific samples and on hsa br M 2ss.. The quality values are given in the phred-33 format, for all samples.
Raw reads were aligned with TopHat version 1.4.0 and Bowtie version 0.12.5. Command line for TopHat: tophat -p 4 -o -a 8 -i 40 -m 1 -I 1000000 -F 0 --coverage-search --microexon-search.
Splice junctions were detected from TopHat unambiguous read alignments, requiring an anchor size of at least 8bp and at most 1 mismatch per alignment.
The genome-wide read coverage was determined using the "wiggles" utility in TopHat.
Expression levels for Ensembl-annotated genes were estimated with Cufflinks 2.0.0, using all reads, with the multi-read correction embedded in Cufflinks.Cufflinks command line: cufflinks -q -G Transcripts_ProteinCoding.gtf -p 4 --library-type ${librarytype} --multi-read-correct. For strand-specific samples, the library-type was set at fr-secondstrand (batch 1) or fr-firststrand (batch 2), while for non-strand-specific samples the library-type was set at fr-unstranded. For expression level computation, GTF files were retrieved from Ensembl 62, and filtered to retain only transcripts annotated as "protein-coding" for protein-coding genes (all transcripts were kept for other gene biotypes).
Genome_build: hg19, gorGor3, MMUL_1, mm9, monDom5, ornAna1, galGal3, JGI 4.2
Supplementary_files_format_and_content: .FPKM files contain expression levels quantified with Cufflinks; the columns are tab-separated. .bedGraph files contain genome-wide read coverage statistics, in bedGraph format, obtained with the "wiggles" utility in TopHat.
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| Submission date |
Jan 15, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Anamaria Necsulea |
| E-mail(s) |
anamaria.necsulea@univ-lyon1.fr
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| Organization name |
CNRS, Université Claude Bernard Lyon 1
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| Department |
Laboratoire de Biométrie et Biologie Evolutive
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| Street address |
43 Bd. du 11 Novembre 1918
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| City |
Lyon |
| ZIP/Postal code |
69622 |
| Country |
France |
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| Platform ID |
GPL13741 |
| Series (1) |
| GSE43520 |
The evolution of lncRNA repertoires and expression patterns in tetrapods |
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| Relations |
| SRA |
SRX217708 |
| BioSample |
SAMN01886779 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1064860_xtr_kd_m1.FPKM.txt.gz |
535.4 Kb |
(ftp)(http) |
TXT |
| GSM1064860_xtr_kd_m1.bedGraph.gz |
79.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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