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SRP029933
Mus musculus
52 Downloadable Samples
Illumina HiSeq 2000
Description
Transcriptomics data obtained from limiting amounts of mRNA is often noisy, providing primarily qualitative changes in transcript expressions. So far, technical variations arising out of the library preparation protocols have not been adequately characterized at reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, stochasticity in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations, which were sequencing depth independent, ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA. Overall design: Sequencing libraries were prepared from serial dilutions of mRNA, ranging from 1 ng to 25 pg, using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq. The mRNA was derived from an in vitro model of lineage segregation achieved by modulating TGF beta signaling pathway in differentiating mouse embryonic stem cells.
Publication Title
Technical variations in low-input RNA-seq methodologies.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 46 Downloadable Samples
- Illumina HiSeq 2000, Affymetrix Human Transcriptome Array 2.0 (hta20)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 28 Downloadable Samples
Affymetrix Human Transcriptome Array 2.0 (hta20), Illumina HiSeq 2000
Description
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as central interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, peripheral interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
Publication Title
The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 18 Downloadable Samples
- Illumina HiSeq 2000, Affymetrix Human Transcriptome Array 2.0 (hta20)
Description
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as central interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, peripheral interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
Publication Title
The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 10 Downloadable Samples
Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
We used microarrays to detect the differences in gene-expression of the periontal ligament between patients with healthy periodontal ligament and patients with periodontitis
Publication Title
The pathology of bone tissue during peri-implantitis.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 8 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
In this study we want to ascertain the differences and similarities of infected and inflammated peri implant tissue versus healthy peri implant tissue at the mRNA level.
Publication Title
The pathology of bone tissue during peri-implantitis.
Sample Metadata Fields
Specimen part, Disease, Disease stage
View Samples- Ovis aries
- 40 Downloadable Samples
- Ovine Gene 1.1 ST Array (ovigene11st)
Description
Feeding animals with either concentrates supplemented with vitamin E or alfalfa grazing has been proven to reduce the oxidative process that occurs in meat products. Indoor-kept lambs were fed a standard concentrate (n=7, C) or concentrate supplemented with vitamin E (n=7, VE) for 30 days before slaughtering all animals at 2224 kg of live weight. Simultaneously, 7 unweaned lambs grazed in alfalfa paddocks (ALF) with their dams. Global transcriptomic data of longissimus thoracis (LT) muscle and subcutaneous fat (SF) with the Affymetrix Ovine Gene 1.1 microarray was used. In LT muscle when ALF group was compared with C group, were identified 41 genes differentially expressed. Among these genes 32 were down- regulated and 9 were up- regulated. Meanwhile when VE treatment was compared with C group were identified a total of 29 genes, 26 were down- regulated and 3 genes were up- regulated. In SF when ALF treatment was compared with C, were identified only 4 genes differentially expressed, all of them up-regulated in ALF group. Meanwhile when VE treatment was compared with C group, were identified a total of 330 genes. Among them, 295 genes were up- regulated and 35 were down- regulated. In LT muscle the clusters corresponding to gene expression profiles from treatments ALF, C and VE were clearly separated from each other. In SF, ALF group, overlap with VE and C treatments, however, VE and C clearly were separate in different clusters. These differentially expressed genes were selected for a functional analysis by using DAVID. In LT muscle some of the identified significant biological processes were catabolic and lipid process (down-regulated, except CPT1B) (CPT1B, PLA2G16, SPSB1, LRTOMT, PLCD4, FBXO9, CNBP and CYP27A1) and muscle organ differentiation (down-regulated) (CPT1B, MYOD1, MYLK2 and MSTN) in ALF; whereas intracellular signaling cascade (IGF1R, DEF8, AKAP7 and CISH) was down-regulated. In SF, vitamin E supplementation had an important effect; most of the genes were up-regulated. DAVID analysis showed that biosynthesis lipid pathway was the most represented with 20 genes, such as EBP, MVD, CYP51A1, DHCR7, HMGCS1, LSS and FDFT1 implicated in cholesterol synthesis. Further exploration of the links between these genes and vitamin E will lead to a better understanding of how vitamin E affects the oxidative process that occurs in meat products.
Publication Title
Genome-wide expression profiling in muscle and subcutaneous fat of lambs in response to the intake of concentrate supplemented with vitamin E.
Sample Metadata Fields
Sex, Treatment
View Samples- Mus musculus
- 11 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Stem cells for murine interstitial cells of cajal suppress cellular immunity and colitis via prostaglandin E2 secretion.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 391 Downloadable Samples
- NextSeq 500
Description
Human immunity relies on the coordinated responses of many cellular subsets and functional states. Inter-individual variations in cellular composition and communication could thus potentially alter host protection. Here, we explore this hypothesis by applying single-cell RNA-Seq to examine viral responses among the dendritic cells (DCs) of three elite controllers (ECs) of HIV-1 infection. We discover a highly functional antiviral DC state in ECs whose fractional abundance after in vitro exposure to HIV-1 correlates with higher CD4+ T cell counts and lower HIV-1 viral loads, and that effectively primes polyfunctional T cell responses in vitro. We identify and validate select immunomodulators that increase the fractional abundance of this state in primary peripheral blood mononuclear cells (PBMCs) from healthy individuals in vitro. Overall design: Single-cell RNA-seq profiling of HIV-1-exposed cDCs and media controls from 3 elite controllers used to identify reproducible gene expression programs associated with cell-intrinsic HIV-1 immune recognition.
Publication Title
A Reproducibility-Based Computational Framework Identifies an Inducible, Enhanced Antiviral State in Dendritic Cells from HIV-1 Elite Controllers.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 19 Downloadable Samples
- NextSeq 500
Description
Human immunity relies on the coordinated responses of many cellular subsets and functional states. Inter-individual variations in cellular composition and communication could thus potentially alter host protection. Here, we explore this hypothesis by applying single-cell RNA-Seq to examine viral responses among the dendritic cells (DCs) of three elite controllers (ECs) of HIV-1 infection. We discover a highly functional antiviral DC state in ECs whose fractional abundance after in vitro exposure to HIV-1 correlates with higher CD4+ T cell counts and lower HIV-1 viral loads, and that effectively primes polyfunctional T cell responses in vitro. We identify and validate select immunomodulators that increase the fractional abundance of this state in primary peripheral blood mononuclear cells (PBMCs) from healthy individuals in vitro. Overall design: Bulk RNA-seq profiling of sorted cDC subsets associated with cell-intrinsic HIV-1 immune recognition.
Publication Title
A Reproducibility-Based Computational Framework Identifies an Inducible, Enhanced Antiviral State in Dendritic Cells from HIV-1 Elite Controllers.
Sample Metadata Fields
Subject, Time
View Samples