Investigation Title Gene Expression of MATa yeast segregants (YJM789 X S96) after alpha factor treatment Comment[Submitted Name] Gene Expression of MATa yeast segregants (YJM789 X S96) after alpha factor treatment Experimental Design transcription profiling by array Experimental Design Term Source REF EFO Comment[SecondaryAccession] GSE19634 Comment[ArrayExpressReleaseDate] 2010-05-14 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-GEOD-19634 Comment[MAGETAB TimeStamp_Version] 2010-08-04 02:57:03 Last Changed Rev: 13058 Experimental Factor Name TREATMENT Experimental Factor Type treatment Experimental Factor Term Source REF Person Last Name Mancera Zhao Steinmetz Snyder Zheng Person First Name Eugenio Hongyu Lars Michael Wei Person Mid Initials Person Email wei.zheng@yale.edu Person Phone Person Fax Person Address Yale University, Suite 503, 300 George Street, New Haven, CT, USA Person Affiliation Yale University Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-05-14 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description In this study, we mapped for the first time differences in transcription binding among individuals and elucidated the genetic basis of such variation. Whole-genome Ste12 binding profiles were determined using ChIP-Seq in pheromone-treated cells of 43 segregants of a cross between two highly diverged yeast strains, YJM789 and S288c, as well as the parental lines. We identified extensive Ste12 binding variation among individuals and mapped underlying cis- and trans- acting loci responsible for such variation. We showed that the majority of TF binding variation is cis-linked and that many variations are associated with polymorphisms residing in the binding motifs of Ste12 as well as those of several known and proposed Ste12 cofactors. We also identified two trans factors, AMN1 and FLO8, that modulate Ste12 binding to promoters of more than 10 genes under α-factor treatment. Neither of these two genes was known to regulate Ste12 previously, and we suggest that they may be key mediators of gene activity and phenotypic diversity. Ste12 binding strongly correlates with gene expression for more than 200 genes, indicating that binding variation is functional. Many of the variable bound genes are involved in cell wall organization and biogenesis. Overall, we identified key regulators of molecular diversity among individuals and provide novel insights into mechanisms of gene regulation. We measured gene expression levels after 30 minutes treatment with alpha factor for 43 MATa segregants from a YJM789 X S96 cross, as well as MATa parental lines. We also measured the gene expression levels without alpha factor treatment for parental lines (S96, HS959) and SEG8 as controls. Protocol Name P-GSE19634-2 P-GSE19634-9 P-GSE19634-4 P-GSE19634-3 P-GSE19634-5 P-GSE19634-6 P-GSE19634-7 P-GSE19634-8 P-GSE19634-1 Protocol Type specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction grow labeling hybridization image_aquisition feature_extraction bioassay_data_transformation Protocol Description Incubation at 30C for 30 minutes with 200rpm swirling, in the presence of 5uM alpha factor. Incubation at 30C for 30 minutes with 200rpm swirling. Ambion Ribopure-yeast kit was used to extract total RNA from 10 ml pheromone-treated yeast cultures, or untreated cultures as control. The RNA concentration was measured using a NanoDrop system. RNA integrity was examined by Agilent Bioanalyzer, and all samples had 260/280 ratios around 2.2, and 260/230 ratios larger than 2.0. Yeast cells were grown in 500ml YPAD medium to mid-log phase (O.D. = 0.6). Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. 0.8 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Yeast Gene Expression 8x15K Microarrays (design ID: 016322) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation. Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%). The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background-subtracted and spatially detrended Processed Signal intensities. ID_REF =
VALUE = Processed Cy3 signal intensity Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology SDRF File E-GEOD-19634.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version