We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish. Overall design: Differential expression analysis of mRNA levels in a single time-point (24 hpf) between wild-type and Tbx2b deficient FAC sorted pineal complex cells
Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing.
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View SamplesWe compared polyIC stimulated cells in the presence of either GFP or NS5 protein Overall design: A549 cells presence with GFP- or NS5-expressing plasmid using Polyjet (Signagen) according to manufacturer instructions, and 30 hours later stimulated with polyIC (Tocris) as previously described (Marazzi et al., 2012). At 12 hours post-stimulation, total cellular RNA was purified by RNeasy column (Qiagen).
Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.
Specimen part, Cell line, Subject
View SamplesU2AF65 is an essential splicing factor involved in the 3'splice site recognition dureing the first steps of spliceosome assembly. In addition, this protein has nucleocytoplasmic shuttling activity and the Drosophila homologue has been implicated in mRNA export.
Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors.
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View SamplesPTB is multifunctional RNA binding protein reported to be involved in splicing, 3' -end processing, stability and translational regulation.
Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors.
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View SamplesOverall goal: To identify genes that will cause non-fusogenic fibroblasts to become fusogenic. Purpose of analysis: To generate transcriptional profile of non-fusogenic fibroblasts, using 10T1/2 fibroblasts transduced with empty retrovirus as model. Experimental structure: The profile generated from the RNAseq analysis would be compared with transcriptional profile of MyoD-expressing fibroblasts (GEO DataSet GSE34907) to identify genes regulating fusion in muscle cells. Overall design: RNAseq analysis of total RNA from 10T1/2 fibroblasts transduced with retrovirus carrying empty pBabe-X retroviral vector was carried out to generate a transcriptional profile of a model of non-fusogenic fibroblasts.
Myomerger induces fusion of non-fusogenic cells and is required for skeletal muscle development.
Specimen part, Cell line, Subject
View SamplesGenome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with diseases of the colon including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). However, the functional role of many of these SNPs is largely unknown and tissue-specific resources are lacking. Expression quantitative trait loci (eQTL) mapping identifies target genes of disease-associated SNPs. Here, we comprehensively map eQTLs in the human colon, assess their relevance for GWAS of colonic diseases and provide functional characterization.
Enrichment of inflammatory bowel disease and colorectal cancer risk variants in colon expression quantitative trait loci.
Sex, Specimen part
View SamplesThis SuperSeries is composed of the SubSeries listed below.
COLOMBOS v2.0: an ever expanding collection of bacterial expression compendia.
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View SamplesGene expression from Escherichia coli.
COLOMBOS v2.0: an ever expanding collection of bacterial expression compendia.
No sample metadata fields
View SamplesNuclear receptor subfamily 1, group D, member 1 (Nr1d1) (also known as Rev-erb alpha) has been linked to circadian rhythm regulation, mood-related behavior, and disorders associated with social deficits. Recent work from our laboratory found striking decreases in Nr1d1 in nucleus accumbens (NAc) in the maternal condition and indirect evidence that Nr1d1 was interacting with numerous addiction and reward-related genes to modulate social reward. In this study, we applied our insights from the maternal state to non-parental adult mice to determine whether decreases in Nr1d1 expression in NAc via adeno-associated viral (AAV) vectors and short hairpin RNA (shRNA)-mediated gene knockdown were sufficient to modulate social reward and mood-related behaviors. We also used microarray analysis of to identify gene expression alterations induced by the lowering of Nr1d1 expression.
The circadian gene Nr1d1 in the mouse nucleus accumbens modulates sociability and anxiety-related behavior.
Sex, Age, Specimen part
View SamplesCancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared to single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared to single agents. Mice, bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared to single agents. A set of 37 genes cooperatively affected (34 down-regulated; 3 up-regulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes down-regulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared to normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo.
Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation.
No sample metadata fields
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