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| Status |
Public on Dec 01, 2009 |
| Title |
Human male 1 rep1 |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Homo sapiens |
| Characteristics |
sex: male
tissue: liver
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
We extracted RNA from each tissue sample using Trizol (Invitrogen, Carisbad, CA), and confirmed that the RNA was of high quality both by visualizing the RNA on a gel, and by analyzing it using Agillent’s Bioanalyzer 2100. We then prepared samples for RNA sequencing using Illumina’s technology (Solexa) by using our previously published RNAseq protocol (Marioni et al., 2008; detailed protocol is available at http://giladlab.uchicago.edu). This method involves several steps that are designed to convert total RNA into a library of template molecules suitable for high throughput DNA sequencing. The first step involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and a high concentration of random hexamer primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. Finally the short cDNA fragments are prepared for sequencing on the Illumina Genome Analyzer using reagents provided in the Genomic DNA Sequencing Sample Prep Kit available with the system. A full and detailed protocol is available at http://giladlab.uchicago.edu.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
n/a
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| Data processing |
In order to compare exon and gene expression levels across species, we used BLAT to identify human exons for which clear orthologs exist in the other two species. To avoid biases due to mapping problems, we removed exons for which multiple plausible orthologs or highly similar paralogs exist. This resulted in the identification of 150,108 orthologous exons in 20,689 genes. We then used MAQ to align reads to their corresponding genome sequences (human: hg18, March 2006 draft; chimpanzee: panTro2, March 2006 draft; rhesus macaque: rheMac2, January 2006 draft) and counted the number of reads that mapped to orthologous exons in each sample. We excluded reads that (i) did not overlap any of the three-species orthologous exons, (ii) had a MAQ mapping quality lower than 20, which might indicate errors or ambiguous mapping, or (iii) mapped to more than a single exon in our list. The number of reads mapped to each gene in each lane is available in the processed data file (ReadCountPerLane.txt).
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| Submission date |
Jul 23, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Ran Blekhman |
| Organization name |
University of Chicago
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| Street address |
920 E. 58th St.
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE17274 |
Sex-specific and lineage-specific alternative splicing in primates |
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| Relations |
| SRA |
SRX014824 |
| BioSample |
SAMN00007010 |
| 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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