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Sample GSM432600 Query DataSets for GSM432600
Status Public on Dec 01, 2009
Title Human female 2 rep1
Sample type SRA
 
Source name liver
Organism Homo sapiens
Characteristics sex: female
tissue: liver
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
 
Description n/a
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).
 
Submission date Jul 23, 2009
Last update date May 15, 2019
Contact name Ran Blekhman
Organization name University of Chicago
Street address 920 E. 58th St.
City Chicago
State/province IL
ZIP/Postal code 60637
Country USA
 
Platform ID GPL9115
Series (1)
GSE17274 Sex-specific and lineage-specific alternative splicing in primates
Relations
SRA SRX014820
BioSample SAMN00007006

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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