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SRP078918
Mus musculus
88 Downloadable Samples
Illumina HiSeq 2500
Description
Regulatory T (Treg) cells, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. This feature implied a key role for a simple network based on IL-2 consumption by Treg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the Treg lineage specification factor Foxp3, confounding experimental efforts to understand the role of IL-2R expression and signaling in Treg suppressor function. Using genetic gain and loss of function approaches, we demonstrate that IL-2 capture is dispensable for control of CD4+ T cells, but is important for limiting CD8+ T cell activation, and that IL-2R dependent STAT5 transcription factor activation plays an essential role in Treg suppressor function separable from T cell receptor signaling. Overall design: Gene expression profiles in Treg cells with or without an expression of an active form of STAT5 were compared by RNA sequencing. Male 8-wk-old Foxp3Cre-ERT2Rosa26Stat5bCA (STAT5b-CA) and Foxp3Cre-ERT2 (control) mice, nine mice for each experimental group, received a single dose (4 mg) of tamoxifen by oral gavage 4 months before isolation. Splenic CD4+Foxp3(YFP/GFP)+GITRhiCD25hi Treg and CD4+Foxp3(YFP/GFP)-CD62LhiCD44lo T naive cells were double sorted using a BD FACSAria II cell sorter. The T cell subsets isolated from three individual mice in the same experimental group (genotype) was pooled into one biological replicate; three biological replicates were generated. A total of 12 samples, i.e., two genotypes, two cell cypes, and three replicates, was subjected to RNA-seq analysis. Samples were sequenced on the Illumina HiSeq 2500 to an average depth of 27.5 million 50-bp read pairs per sample.
Publication Title
Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function.
Sample Metadata Fields
Sex, Specimen part, Subject
View Samples- Gallus gallus
- 94 Downloadable Samples
- Illumina HiSeq 2500
Description
The pituitary gland is a neuroendocrine organ that is involved in several processes within the body such as metabolism, growth, immune function, and reproduction. Increased ambient temperatures are environmental stressor that leads to several welfare concerns in poultry production but also economic losses. Because of the involvement of the pituitary gland in several processes that are affected by heat stress, it is hypothesized this tissue''s gene expression will be impacted by heat stress. The objectives of the project are to (a) identify genes that constitue the pituitary gland when compared to other collected chicken tissues (Insert tissues) and (b) identify genes that respond to heat stress via differential expression analysis to better understand the chicken''s response to heat at the transcriptomic level. Overall design: Ross 708 broiler chickens were raised from day of hatch to day 42, typical market age, on the University of Delaware farm. Birds were placed into two separate houses, one thermoneutral house and one experimental (heat stress) house. Both houses were kept at 23 hours of light and 1 hour of dark and birds were placed on litter and given feed (meeting all NRC requirements) and water with ad libitum access. Both houses were kept at 35 degrees celsius for the first week and the temperature was decreased 5 degrees celsius each week until 25 degrees celsius. The thermoneutral hosue was maintained at 25 degrees celsius for the remainder of the study. Starting on day 21, the experimental house began a cyclical heat stress scheme with 8 hours per day of increased temperatures (35 - 37 degrees celsius) through completion of the trial at day 42. Necropsies were performed at several points throughout the trial (days 21, 22, 26, 32, and 42).
Publication Title
Transcriptomic changes throughout post-hatch development in Gallus gallus pituitary.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 187 Downloadable Samples
Illumina HumanHT-12 V3.0 expression beadchip
Description
Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene regulation. We found a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene regulatory programs in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. As expected, previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that findings and insight drawn from gene regulatory studies in mature LCLs are generally not affected by artificial nature of the LCL model system and are likely to faithfully reflect regulatory interactions in primary tissues. However, our data indicate that many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.
Publication Title
The effect of freeze-thaw cycles on gene expression levels in lymphoblastoid cell lines.
Sample Metadata Fields
Sex, Specimen part
View Samples- Rattus norvegicus
- 8 Downloadable Samples
- Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
In the growth plate, the reserve and perichondral zones have been hypothesized to have similar functions, but their exact functions are poorly understood. Our hypothesis was that significant differential gene expression exists between perichondral and reserve chondrocytes that may differentiate the respective functions of these two zones. Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC), reserve zone (RZ), proliferative zone (PZ), and hypertrophic zone (HZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator.
Publication Title
Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 5 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
During pregnancy, cells from each fetus travel into the maternal circulation and organs, resulting in the development of microchimerism. Identification of the cell types in this microchimeric population would permit better understanding of possible mechanisms by which they affect maternal health. However, comprehensive analysis of fetal cells has been hampered by their rarity. In this study, we sought to overcome this obstacle by combining flow cytometry with multidimensional gene expression microarray analysis of fetal cells isolated from the murine maternal lung during late pregnancy. Fetal cells were collected from the lungs of pregnant female mice. cDNA was amplified and hybridized to gene expression microarrays. The resulting fetal cell core transcriptome was interrogated using multiple methods including Ingenuity Pathway Analysis, the BioGPS gene expression database, principal component analysis, the Eurexpress gene expression atlas and primary literature. Here we report that small numbers of fetal cells can be flow sorted from the maternal lung, facilitating discovery-driven gene expression analysis. We additionally show that gene expression data can provide functional information about the fetal cells. Our results suggest that fetal cells in the murine maternal lung are a mixed population, consisting of trophoblasts, mesenchymal stem cells and cells of the immune system. The detection of trophoblasts and immune cells in the maternal lung may facilitate future mechanistic studies related to the development of immune tolerance and pregnancy-related complications, such as preeclampsia. Furthermore, the presence and persistence of mesenchymal stem cells in maternal organs may have implications for long-term postpartum maternal health.
Publication Title
Comprehensive analysis of genes expressed by rare microchimeric fetal cells in the maternal mouse lung.
Sample Metadata Fields
No sample metadata fields
View Samples- Arabidopsis thaliana
- 5 Downloadable Samples
- Affymetrix Arabidopsis ATH1 Genome Array (ath1121501)
Description
The aim of this study is to identify Arabidopsis genes whose expression is altered by aphid feeding. An understanding of the plant aphid interaction at the level of the plant transcriptome will 1) consolidate current areas of investigation focused on the phloem composition (the aphid diet), 2) open up areas of plant aphid interactions for ourselves and other workers, 3) Contribute to understanding the use of new molecular technologies in an environmental context and 4) contribute to existing and development of novel control strategies.Our Arabidopsis/Myzus persicae system provides a valuable model for the study because of: a) the advantages of using Arabidopsis, b) The ability to use clonal insects, c) phloem feeding aphids facilitate focus on a specific cell type, d) aphid stylectomy allows collection of pure phloem sap to monitor phloem phenotype of the plant and the insect diet, e) we have techniques to monitor the reproductive performance and feeding behaviour aphids.Our strategy has been to test the function of selected genes, particularly those regulating phloem composition (the feeding site of the aphid) based on current phloem models of phloem function. Gene choice is limited the simplicity of current models of phloem aphid interaction.We propose a simple two treatment (aphid infested vs control plants) experiment that will identify novel target genes for future analysis. Arabidopsis plants (variety Columbia) will be grown in 16/8 light/dark in temperature controlled growth rooms. At growth stage 3.90, when rosette growth is complete, 10 clonal adult Myzus persicae will be caged in clip cages on the two largest leaves on each plant. Control plants will be treated identically except that the cages will be empty. Leaves will be harvested 8 h after infestation. This time point is selected as we know that 90% of aphids are plugged into the sieve element within 2h and that a 6h lag phase has period has previously been used when examining gene expression affected by wounding. In subsequent experiments we will examine time courses of expression of relevant genes using other approaches. Pooling two leaves from each of ten plants will generate the RNA sample, ensuring that expression signals are representative of the population of plants.
Publication Title
Exploring plant responses to aphid feeding using a full Arabidopsis microarray reveals a small number of genes with significantly altered expression.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 71 Downloadable Samples
- IlluminaHiSeq2500
Description
Genetic variants that impact gene regulation are important contributors to human phenotypic variation. For this reason, considerable efforts have been made to identify genetic associations with differences in mRNA levels of nearby genes, namely, cis expression quantitative trait loci (eQTLs). The phenotypic consequences of eQTLs are presumably due, in most cases, to their ultimate effects on protein expression levels. Yet, only few studies have quantified the impact of genetic variation on proteins levels directly. It remains unclear how faithfully eQTLs are reflected at the protein level, and whether there is a significant layer of cis regulatory variation acting primarily on translation or steady state protein levels. To address these questions, we measured ribosome occupancy by high-throughput sequencing, and relative protein levels by high-resolution quantitative mass spectrometry, in a panel of lymphoblastoid cell lines (LCLs) in which we had previously measured transcript expression using RNA sequencing. We then mapped genetic variants that are associated with changes in transcript expression (eQTLs), ribosome occupancy (rQTLs), or protein abundance (pQTLs). Most of the QTLs we detected are associated with transcript expression levels, with consequent effects on ribosome and protein levels. However, we found that eQTLs tend to have significantly reduced effect sizes on protein levels, suggesting that their potential impact on downstream phenotypes is often attenuated or buffered. Additionally, we confirmed the presence of a class of cis QTLs that specifically affect protein abundance with little or no effect on mRNA levels; most of these QTLs have little effect on ribosome occupancy, and hence may arise from differences in post-translational regulation. Overall design: We measured level of translation transcriptome-wide in lymphoblastoid cell lines derived from 72 HapMap Yoruba individuals using ribosome profiling assay, for which we have transcript level, protein level (62 out of 72) and genotype information collected.
Publication Title
Genomic variation. Impact of regulatory variation from RNA to protein.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 30 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Stem cell differentiation is known to involve changes in RNA expression, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of embryonic stem cells (ESCs) into embyroid bodies (EBs) by integrating conventional transcriptome analysis with global assessment of ribosome loading. Differentiation was accompanied by an anabolic switch, characterized by global increases in transcript abundance, polysome content, protein synthesis rates and protein content. Furthermore, 78% of expressed transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Elevated protein synthesis was accompanied by enhanced phosphorylation of eIF-4E binding protein, suggesting regulation by the mTOR pathway.
Publication Title
A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 4 Downloadable Samples
- IlluminaGenomeAnalyzerII
Description
Next-generation sequencing has become an important tool for genome-wide quantification of DNA and RNA. However, a major technical hurdle lies in the need to map short sequence reads back to their correct locations in a reference genome. Here we investigate the impact of SNP variation on the reliability of read-mapping in the context of detecting allele-specific expression (ASE).We generated sixteen million 35 bp reads from mRNA of each of two HapMap Yoruba individuals. When we mapped these reads to the human genome we found that, at heterozygous SNPs, there was a significant bias towards higher mapping rates of the allele in the reference sequence, compared to the alternative allele. Masking known SNP positions in the genome sequence eliminated the reference bias but, surprisingly, did not lead to more reliable results overall. We find that even after masking, $\sim$5-10\% of SNPs still have an inherent bias towards more effective mapping of one allele. Filtering out inherently biased SNPs removes 40\% of the top signals of ASE. The remaining SNPs showing ASE are enriched in genes previously known to harbor cis-regulatory variation or known to show uniparental imprinting. Our results have implications for a variety of applications involving detection of alternate alleles from short-read sequence data. Scripts, written in Perl and R, for simulating short reads, masking SNP variation in a reference genome, and analyzing the simulation output are available upon request from JFD. Overall design: RNA-Seq on two YRI Hapmap cell lines. Each individual sequenced on two lanes of the Illumina Genome Analyzer
Publication Title
Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 372 Downloadable Samples
- IlluminaHiSeq2500
Description
Noncoding variants play a central role in the genetics of complex traits, but we still lack a full description of the main molecular pathways through which they act. Here we used molecular data to quantify the contribution of cis-acting genetic effects at each major stage of gene regulation from chromatin to proteins, within a population sample of Yoruba lymphoblastoid cell lines (LCLs). We performed 4sU metabolic labeled transcripts in 65 YRI LCLs to identify genetic variants that affect transcription rates. As expected, we found an important contribution of genetic variation via chromatin, contributing ~65% of eQTLs (expression Quantitative Trait Loci). The remaining eQTLs, which are not asso- ciated with chromatin-level variation, are highly enriched in transcribed regions, and hence may affect expression through co- or post-transcriptional processes. Overall design: International HapMap lymphoblastoid cell lines (LCLs) derived from YRI (Yoruba in Ibadan, Nigeria); We adapted the 4sU labelling method from (PMID 21516085). Briefly, cell cultures were grown to log phase in volumes sufficient to yield about 300 ng of 4sU-labeled RNA. Cells were incubated with 4sU for the required length of time (0, 30, or 60 minutes), then washed, pelleted, and frozen. Total RNA was extracted, and 4sU-labeled RNA was separated from total RNA using a bead-based biotin-streptavidin purification protocol. We sequenced metabolic labeled transcripts in 65 YRI LCLs 30 minutes and 60 minutes after incubation.
Publication Title
RNA splicing is a primary link between genetic variation and disease.
Sample Metadata Fields
No sample metadata fields
View Samples