Single-cell expression profiling is a rich resource of cellular heterogeneity. While profiling every sample under study is advantageous, such workflow is time consuming and costly. We devised CPM - a deconvolution algorithm in which cellular heterogeneity is inferred from bulk expression data based on pre-existing collection of single-cell RNA-seq profiles. We applied CPM to investigate individual variation in heterogeneity of murine lung cells during in vivo influenza virus infection, revealing that the relations between cell quantities and clinical outcomes varies in a gradual manner along the cellular activation process. Validation experiments confirmed these gradual changes along the cellular activation trajectory. Additional analysis suggests that clinical outcomes relate to the rate of cell activation at the early stages of this process. These findings demonstrate the utility of CPM as a mapping deconvolution tool at single-cell resolution, and highlight the importance of such fine cell landscape for understanding diversity of clinical outcomes. Overall design: Lungs gene expression of Collaborative Cross mice taken 48h after the infection with either the influenza virus or PBS.
Cell composition analysis of bulk genomics using single-cell data.
Specimen part, Subject, Time
View SamplesPrimary human foreskin fibroblasts (HFF) were exposed to either salt stress (80mM KCl) or heat stress (44ºC). Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013. Overall design: Newly transcribed RNA was labelled in one hour intervals during either salt or heat stress (prior to stress, 0-1h or 1-2h). All 4sU-RNA samples were sent for sequencing. Two independent biological replicates were analysed.
HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes.
Specimen part, Subject, Time
View SamplesQuiescent stem cells of glioblastoma (GBM), a malignant primary brain tumor, are potential sources for recurrence after therapy. However, the gene expression program underlying the physiology of GBM stem cells remains unclear. We have isolated quiescent GBM cells by engineering them with a knock-in H2B-GFP proliferation reporter and expanding them in a 3D tumor organoid model that mimics tumor heterogeneity. H2B-GFP label retaining quiescent cells were subjected to stem cell assays and RNA-Seq gene expression analysis. While quiescent GBM cells were similar in clonal culture assays to their proliferative counterparts, they displayed higher therapy resistance. Interestingly, quiescent GBM cells upregulated epithelial-mesenchymal transition (EMT) genes and genes of extracellular matrix components. Our findings connect quiescent GBM cells with an EMT-like shift, possibly explaining how GBM stem cells achieve high therapy resistance and invasiveness, and suggest new targets to abrogate GBM. Overall design: Glioblastoma cancer cells in 3D organoid culture were pulsed for 2 weeks with H2B-GFP, then chased either 2 or 4 weeks. Label-retaining GFP-high cells (quiescent) were separated from bulk population, and both populations were analyzed by RNA-Seq.
Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment.
Specimen part, Subject
View SamplesPrimary human foreskin fibroblasts (HFF) were infected with wild-type simplex virus 1 (HSV-1) strain 17 at a multiplicity of infection (MOI) of 10. Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013. Overall design: Newly transcribed RNA was labelled in one hour intervals during the first eight hours of HSV-1 infection. All nine 4sU-RNA samples as well as total cellular RNA of every second hour of infection were sent for sequencing. Two independent biological replicates were analysed.
Prediction of Poly(A) Sites by Poly(A) Read Mapping.
No sample metadata fields
View SamplesThis SuperSeries is composed of the SubSeries listed below.
PDEF promotes luminal differentiation and acts as a survival factor for ER-positive breast cancer cells.
Cell line, Treatment
View SamplesRecent studies suggest that PDEF is required for secretory cell differentiation in several epithelial tissues. To investigate PDEF in the mammary gland, we examined the effect of this transcription factor on gene expression using microarray based profiling of MCF-10A cells. These cells are non-transformed mammary epithelial cells that express protein and gene expression programs of basal epithelial cells and undetectable levels of endogenous PDEF. Bioinformatics analysis of the genes induced or repressed by PDEF overexpression in MCF10A cells revealed a striking effect on expression of luminal and myoepithelial cell markers.
PDEF promotes luminal differentiation and acts as a survival factor for ER-positive breast cancer cells.
Cell line, Treatment
View SamplesMicroarray gene expression analysis was performed in MCF7 cells transduced with a non-specific shRNA or PDEF-targeting shRNA, and both subjected to hormone depletion for 48 hours. Analyses of differentially expressed genes combined with gene ontology revealed a downregulation of cell cycle related-genes and an upregulation of apoptosis-related genes in PDEF knockdown cells. These target genes constitute potential effectors of the pro-survival role of PDEF.
PDEF promotes luminal differentiation and acts as a survival factor for ER-positive breast cancer cells.
Cell line, Treatment
View SamplesPresbycusis age-related hearing loss is the number one communicative disorder of our aged population. Here we analyzed gene expression for a set of GABA receptors in the cochlea of aging CBA mice using the Affymetrix GeneChip MOE430A. Functional phenotypic hearing measures distortion-product otoacoustic emission (DPOAE) amplitudes (four age groups) were made. The gene expression changes from RMA normalized microarray data (40 replicates) were first subjected to one-way ANOVA, and then linear regression was performed. In addition, the log signal ratio was converted to fold change, and selected gene expression changes were confirmed by relative real-time PCR. Major findings: expression of GABA-A receptor subunit 6was upregulated with age and hearing loss, whereas subunit 1 was repressed. In addition, GABA-A receptor associated protein like-1 and GABA-A receptor associated protein like-2 were strongly downregulated with age and hearing impairment. Lastly, gene expression measures were correlated with pathway/network relationships relevant to the inner ear using Pathway Architect, to identify key pathways consistent with the gene expression changes observed.
Novel approach to select genes from RMA normalized microarray data using functional hearing tests in aging mice.
Sex
View SamplesWe applied RNA-seq analysis to human islet cells, received from 3 independent donors, treated with either redifferentiation cocktail + ARX shRNA, or redifferentiation cocktail + control shRNA or left untreated. Overall design: Examination of the effect of ARX inhibition on redifferentiation of ß-cell-derived (BCD) cells
Redifferentiation of expanded human islet β cells by inhibition of ARX.
No sample metadata fields
View SamplesThis is an initial experiment which was performed in order to identify novel transcriptional targets of the tumor suppressor p53
p53 activates the PANK1/miRNA-107 gene leading to downregulation of CDK6 and p130 cell cycle proteins.
Specimen part, Cell line, Treatment
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