Comment[ArrayExpressAccession] E-GEOD-17274 MAGE-TAB Version 1.1 Public Release Date 2009-12-01 Investigation Title Sex-specific and lineage-specific alternative splicing in primates Comment[Submitted Name] Sex-specific and lineage-specific alternative splicing in primates Experiment Description Comparative studies of gene regulation suggest an important role for natural selection in shaping gene expression patterns within and between species. Most of these studies, however, estimated gene expression levels using microarray probes designed to hybridize to only a small proportion of each gene. Here we used recently-developed RNA sequencing protocols, which side-step this limitation, to assess intra- and inter-species variation in gene regulatory processes in considerably more detail than was previously possible. Specifically, we used RNAseq to study transcript levels in humans, chimpanzees, and rhesus macaques, using liver RNA samples from three males and three females from each species. Our approach allowed us to identify a large number of genes whose expression levels likely evolve under natural selection in primates. These include a subset of genes with conserved sexually dimorphic expression patterns across the three species, which we found to be enriched for genes involved in lipid metabolism. Our data also suggest that while alternative splicing is tightly regulated within and between species, sex-specific and lineage-specific changes in the expression of different splice forms are also frequent. Intriguingly, among genes in which a change in exon usage occurred exclusively in the human lineage, we found an enrichment of genes involved in anatomical structure and morphogenesis, raising the possibility that differences in the regulation of alternative splicing have been an important force in human evolution. Keywords: Gene Regulation Study Examination of gene expression levels in livers from three primate species (human, chimpanzee, and rhesus macaque), using 3 male and 3 female samples from each species. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Blekhman Blekhman Marioni Zumbo Stephens Gilad Person First Name Ran Ran John Paul Matthew Yoav Person Mid Initials C Person Email geo@ncbi.nlm.nih.gov Person Affiliation University of Chicago Person Address University of Chicago, 920 E. 58th St., Chicago, IL, USA Person Roles submitter Protocol Name P-GSE17274-2 P-GSE17274-1 Protocol Description 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). 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. Protocol Type normalization data transformation protocol nucleic acid library construction protocol Experimental Factor Name SEX ORGANISM Experimental Factor Type sex organism Publication Title Sex-specific and lineage-specific alternative splicing in primates. Publication Author List Blekhman R, Marioni JC, Zumbo P, Stephens M, Gilad Y PubMed ID 20009012 Publication DOI 10.1101/gr.099226.109 Comment[SecondaryAccession] GSE17274 Comment[GEOReleaseDate] 2009-12-01 Comment[ArrayExpressSubmissionDate] 2009-07-23 Comment[GEOLastUpdateDate] 2013-07-01 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE17274_ReadCountPerLane.txt Comment[SecondaryAccession] SRP001558 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR032116-SRR032151 SDRF File E-GEOD-17274.sdrf.txt