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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 26, 2013 |
| Title |
wild.type.10.rnaseq |
| Sample type |
SRA |
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| Source name |
testes
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| Organism |
Mus musculus |
| Characteristics |
developmental stage: 10.5 dpp
strain: C57BL/6J
genotype: wild type
tissue: testes
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were maintained and used according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.
Strand-specific RNA-seq libraries via Incorporation of dUTP in second strand synthesis (Zhang et al., 2012) using Ribo-Zero Gold (Epicentre Biotechnologies, Madison, WI, USA) were sequenced using the paired-end protocol on a HiSeq 2000.
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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 |
De novo transcriptome assembly from three biological replicates of strand-specific RNA-seq data from adult testes was performed using Trinity (r2012-06-08) with default parameters (Grabherr et al., 2011). The assembled RNA sequences were aligned to the mouse genome (mm9) with BLAT (Kent, 2002), and the alignments with more than 95% of sequence length mapped and fewer than 1% mismatches retained. We extracted novel junctions from Trinity (i.e., reads with [0-9]+M[0-9]+N[0-9]+M pattern in the CIGAR string of SAM output), and re-mapped all RNA-seq reads to these junctions, rescuing 1,402,444 reads in three replicates. Rescued reads were combined with TopHat alignments (supplied with "–max-multihits 100" to assembly through repetitive regions) and used as input for reference-based assembly. We used Cufflinks v2.0.2 (Trapnell et al., 2010) with parameters of “-u -j 0.2 --min-frags-per-transfrag 40” to assemble transcripts. To join small transcript fragments caused by insufficient read coverage or embedded repetitive elements, two different gap-joining distance cutoffs were used for the assembly of genes (“--overlap-radius 100”) and piRNA loci (“--overlap-radius 250”). We used Cuffcompare v2.0.2 (Trapnell et al., 2010) to annotate the 49,840 Cufflinks-assembled transcripts using parameters optimized for genic conditions (“--overlap-radius 100”). A final list of 50,081 transcripts (including correct piRNA loci) are provided here.
RNA-seq reads were aligned to the genome (NCBI 37/mm9) using TopHat 2.0.4 (Trapnell et al., 2009). Reads were mapped uniquely using the ‘-g 1’ switch. We assembled the mouse testes transcriptome. For genes with multiple isoforms, the transcript with the highest average rpkm value among the three replicates of adult testes was selected for further analysis. Fragments with both reads mapped within a transcript, or to piRNA precursor transcripts, were counted using BEDTools (Quinlan and Hall, 2010). The sum of the reads aligning to the top quartile of expressed transcripts per library was used to calibrate the samples. The number of reads per transcript was normalized by length, divided by the library-specific calibration factor, and reported as rpkm with a pseudo count of 0.001.
Genome_build: mm9
Supplementary_files_format_and_content: bed file containing assembled transcripts and csv file containing rpkm values available on Series record
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| Submission date |
Feb 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Christian Roy |
| Organization name |
Umass Med
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| Street address |
364 Plantation St
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| City |
Worcester |
| State/province |
Massachusetts |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE44654 |
An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes (RNA-Seq) |
| GSE44690 |
An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes |
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| Relations |
| Reanalyzed by |
GSE80793 |
| SRA |
SRX245674 |
| BioSample |
SAMN01925335 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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