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| Status |
Public on Jan 19, 2014 |
| Title |
mdo pl I 2 [batch2] |
| Sample type |
SRA |
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| Source name |
Placenta
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| Organism |
Monodelphis domestica |
| Characteristics |
Sex: NA
strain: NA
age: 14 days post-conception
extract_protocol: Illumina HiSeq 2000, strand-specific
library-type (for tophat and cufflinks): fr-firststrand
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| Extracted molecule |
total 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 HiSeq 2000 |
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| Description |
polyA RNA
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| Data processing |
The basecalling was performed with the Illumina GAIIx pipeline, version 1.8, as well as with the Illumina HiSeq 2000 pipeline and with Ibis (Kircher et al, 2009). Ibis was used on non-strand specific samples generated with Illumina GAII. 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).
Paired-end RNA-seq samples were treated as single-end for comparability, i.e. only the first read of the pair was used for expression analyses.
Small RNA-seq reads were trimmed to remove adapter sequences. We kept reads with trimmed sizes between 15 and 25 bp, which are compatible with mature miRNA sizes. The trimmed reads were mapped on the Ensembl 62 miRNA precursors, using Bowtie. miRNA expression levels were computed as the number of reads that mapped uniquely to each Ensembl-annotated miRNA precursor.
Genome_build: hg19, mm9, monDom5, ornAna1, galGal3, JGI 4.2
Supplementary_files_format_and_content: The .FPKM files contain expression levels quantified with Cufflinks; the columns are tab-separated.
Supplementary_files_format_and_content: The .bedgraph files contain genome-wide read coverage statistics, obtained with the "wiggles" utility in TopHat.
Supplementary_files_format_and_content: For miRNA data, the .txt files contain the number of reads (unique and total) that could be assigned to a given miRNA precursor annotated in Ensembl. Reads that aligned to more than 1 Ensembl-annotated miRNA precursor were "divided" equally among the different precursors; thus, the "total" number of reads is not necessarily an integer.
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| Submission date |
Jul 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Anamaria Necsulea |
| E-mail(s) |
[email protected]
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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 |
GPL15381 |
| Series (1) |
| GSE43520 |
The evolution of lncRNA repertoires and expression patterns in tetrapods |
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| Relations |
| BioSample |
SAMN02265394 |
| SRA |
SRX328082 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1196051_mdo_pl_I2.FPKM.txt.gz |
571.7 Kb |
(ftp)(http) |
TXT |
| GSM1196051_mdo_pl_I2.bedgraph.gz |
81.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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