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SRP058066
Rattus norvegicus
170 Downloadable Samples
Illumina HiSeq 1000
Description
Purpose: Single-cell whole transcriptome sequencing was used to better understand the mechanism of action of our Dyrk1a inhibitor''s proliferation of pancreatic islets. Methods: primary pancreatic islets were isolated, cultured, and stimulated with either 0.1% DMSO or 3 µM GNF4877. Single cells were captured and cDNA isolated on a Fluidigm C1 instrument. Sequencing libraries were made with Nextera XT reagents (Illumina) and single-end 50 bp reads were generated on an Illumina HiSeq 1000. Reads were mapped to the rat transcriptome. Results: Consistent with GNF4877 eliciting beta cell proliferation, we observed an increase in the number of beta cells co-expressing insulin 1 and genes involved in cell cycle including the M phase marker Cyclin B1. Comparison of Cyclin B1 expressing cells from GNF4877-treated islets to beta cells from DMSO-treated islets further revealed a significant increase expression of genes associated with full cell cycle progression and enrichment of Gene Ontology (GO) categories for proliferation. Conclusions: Since only a small subset of islet cells proliferate when stimulated with GNF4877, single-cell transcriptome sequencing allowed us to examine expression of genes co-regulated with known proliferation markers and will hopefully allow us to characterize beta cell subsets which are responsive to proliferation-associated therapies. Overall design: 84 GNF4877-treated and 86 DMSO-treated rat islet cells containing greater than 100,000 mapped sequencing reads per cell and having a single verified cell per port were compared
Publication Title
Inhibition of DYRK1A and GSK3B induces human β-cell proliferation.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 14 Downloadable Samples
Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Purpose: Klf5 plays a critical role in the mouse ocular surface (Kenchegowda et al., 2011. Dev Biol. 356:5-18). Here, we compare wild-type (WT) and Klf5-conditional null (Klf5CN) corneal gene expression at postnatal day-11 (PN11) and PN56 to identify the Klf5-target genes. Methods: Gene expression was compared using Affymetrix microarrays with QPCR validation. Transient transfection assays examined the effect of Klf5 on selected target gene promoter activities. Whole-mount corneal immunofluorescent staining examined neovascularization and CD45+ macrophage influx. Results: Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases, 72 concordant decreases and 3 discordant changes. Canonical pathway analysis identified 35 and 34 significantly (p<0.001) enriched pathways at PN11 and PN56, respectively, with 24 common pathways. PN56 Klf5CN corneas shared 327 increases and 91 decreases with the previously described Klf4CN corneas (Swamynathan et al., 2008. IOVS 49:3360-70). Angiogenesis and immune response-related genes were affected consistent with lymphangiogenesis and macrophage influx in Klf5CN corneas, respectively. Expression of 1574 genes was increased and 1915 decreased, in the WT PN56 compared with PN11 corneas. Expression of many collagens, matrix metalloproteinases and other extracellular matrix associated genes decreased in WT corneas between PN11 and PN56, while that of solute carrier family members increased. Conclusions: Differences in PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in Klf5 functions during corneal maturation. Klf4- and Klf5-target genes do not overlap, consistent with their non-redundant roles in the mouse cornea.
Publication Title
Critical role of Klf5 in regulating gene expression during post-eyelid opening maturation of mouse corneas.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 10 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Conditional disruption of Klf4 in the ectoderm-derived tissues of the eye results in defective cornea, conjunctiva and the lens.
Publication Title
Regulation of mouse lens maturation and gene expression by Krüppel-like factor 4.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration. Overall design: CRX+ flow sorted cells from human retina derived organoids were collected at 6 time points during differentiation (day (D) 37, 48, 67, 90, 134, 220).
Publication Title
Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.
Sample Metadata Fields
Specimen part, Subject
View Samples- Escherichia coli k-12
- 6 Downloadable Samples
- Affymetrix E. coli Genome 2.0 Array (ecoli2)
Description
The most important approach to the development of platform organisms for recombinant protein production relies on random mutagenesis and phenotypic selection. Complex phenotypes, including those associated with significant elevated expression and secretion of heterologous proteins, are the result of multiple genomic mutations. Using next generation sequencing, a parent and derivative hypersecreter strain (B41) of Escherichia coli were sequenced with an average coverage of 52.8X and 55X, respectively. A new base-pair calling program, revealed a single nucleotide polymorphism in the B41 genome at position 1,074,787, resulting in translation termination near the N-terminus of a transcriptional regulator protein, RutR, coded by the ycdC gene. We verified the hypersecretion phenotype in a ycdC::Tn5 mutant and observed a 3.4-fold increase in active hemolysin secretion, consistent with the increase observed in B41. mRNA expression profiling showed decreased expression of tRNA-synthetases and some amino acid transporters in the ycdC::Tn5 mutant. This study demonstrates that power of next generation sequencing to characterize mutants leading to successful metabolic engineering strategies for strain improvement.
Publication Title
A single nucleotide polymorphism in ycdC alters tRNA synthetase expression and results in hypersecretion in Escherichia coli.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 14 Downloadable Samples
- Illumina HiSeq 2500
Description
Difference in RNA content of different cell types introduces bias to gene expression deconvolution methods. If ERCC spike-ins are introduced into samples, predicted proportions of deconvolution methods can be corrected Overall design: Two cell types of distinctly different sizes and RNA per cell content: HEK cells and Jurkat cells were mixed in different proportions ensuring that each mixture contained total of one million cells. We sequenced RNA of the samples (including ERCC spike-in controls to 382 be able to control for the absolute RNA-concentration).
Publication Title
Complete deconvolution of cellular mixtures based on linearity of transcriptional signatures.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 12 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Purpose: To identify the changes in postnatal mouse conjunctival forniceal gene expression and their regulation by Klf4 around eye opening stage when the goblet cells first appear.
Publication Title
Mouse conjunctival forniceal gene expression during postnatal development and its regulation by Kruppel-like factor 4.
Sample Metadata Fields
No sample metadata fields
View Samples- Danio rerio
- 9 Downloadable Samples
- IlluminaGenomeAnalyzerIIx
Description
Purpose: Zebrafish neurons regenerate from Müller glia following retinal lesions. Genes and signaling pathways important for retinal regeneration in zebrafish have been described, but our understanding of how Mu¨ller glial stem cell properties are regulated is incomplete. Mammalian Mu¨ller glia possess a latent neurogenic capacity that might be enhanced in regenerative therapies to treat degenerative retinal diseases. Methods: To identify transcriptional changes associated with stem cell properties in zebrafish Mu¨ller glia, we performed a comparative transcriptome analysis from isolated cells at 8 and 16 hours following an acute, photic lesion, prior to the asymmetric division that produces retinal progenitors. Results: We report a rapid, dynamic response of zebrafish Müller glia, characterized by activation of pathways related to stress, NF-kappa B signaling, cytokine signaling, immunity, prostaglandin metabolism, circadian rhythm, and pluripotency, and an initial repression of Wnt signaling. When we compared publicly available transcriptomes of isolated mouse Mu¨ller glia from two retinal degeneration models, we found that mouse Müller glia showed evidence of oxidative stress, variable responses associated with immune regulation, and repression of pathways associated with pluripotency, development, and proliferation. Conclusions: Categories of biological processes/pathways activated following photoreceptor loss in regeneration-competent zebrafish Mu¨ller glia, which distinguished them from mouse Mu¨ller glia in retinal degeneration models, included: cytokine signaling (notably NF-kappa B), prostaglandin E2 synthesis, expression of core clock genes, and pathways/metabolic states associated with pluripotency. These regulatory mechanisms are relatively unexplored as potential mediators of stem cell properties likely to be important in Müller glial cells for successful retinal regeneration. Overall design: Transcriptional profiles of 0, 8, and 16 hour post-lesion zebrafish Müller glia (in triplicate) were generated by high-throughput sequencing in an Illumina GAIIx.
Publication Title
Rapid, Dynamic Activation of Müller Glial Stem Cell Responses in Zebrafish.
Sample Metadata Fields
No sample metadata fields
View Samples- Saccharomyces cerevisiae
- 4 Downloadable Samples
- Affymetrix Yeast Genome 2.0 Array (yeast2)
Description
KP1019 (trans-[tetrachlorobis(1H-indazole) ruthenate(III)]) is a ruthenium complex that exhibited anti-cancer activity in several in vitro and in vivo studies. KP1019 was even efficient against cancer cells that were resistant to other chemotherapeutic agents and thus emerged as a promising anti-cancer drug without dose-limiting cytotoxicity. However, the molecular mechanisms of its action are elusive.
Publication Title
A systematic assessment of chemical, genetic, and epigenetic factors influencing the activity of anticancer drug KP1019 (FFC14A).
Sample Metadata Fields
No sample metadata fields
View Samples- Caenorhabditis elegans
- 3 Downloadable Samples
- Affymetrix C. elegans Genome Array (celegans)
Description
Bio-electrospray, the direct jet-based cell handling apporach, is able to handle a wide range of cells. Studies at the genomic, genetic, and the physiological level have shown that, post-treatment, cellular integrity is unperturbed and a high percentage (>70%, compared to control) of cells remain viable. Although, these results are impressive, it may be argued that cell based systems are oversimplistic. This study utilizing a well characterised multicellular model organism, the non-parasitic nematode Caenorhabditis elegans. Nematodes were subjected to bio-electrosprays to demonstrate that bio-electrosprays can be safely applied to nematodes.
Publication Title
Bio-electrospraying the nematode Caenorhabditis elegans: studying whole-genome transcriptional responses and key life cycle parameters.
Sample Metadata Fields
Specimen part
View Samples