We set up a pilot study using Affymetrix Gene Chip Porcine Genome Arrays to evaluate the impact of time lags from death on gene expression profiling of porcine skeletal muscle at four post mortem time points (up to 24 hrs) during the routine processing of fresh tights
Microarray gene expression analysis of porcine skeletal muscle sampled at several post mortem time points.
Sex, Specimen part, Time
View SamplesBET-regulated transcriptome and BRD4, BRD2, BRD3 and Pol II ChIP-seq datasets in human ESCs before and after BET inhibition. Transcription factors and chromatin remodeling complexes are key determinants of embryonic stem cell (ESC) identity. In this study, we investigate the role of BRD4, a member of the bromodomain and extra-terminal domain (BET) family of epigenetic reader proteins, in control of ESC identity. We performed RNA-seq analyiss in the presense of small molecule inhibitors of BET proteins to show that BRD4 positively regulates the ESC transcriptome. We also integrated RNA-seq analysis with ChIP-sequencing datasets s for BRD4 (and for other BRD2 and BRD3) to demonstrate that BRD4 binds SEs and regulates the expression of SE-associated pluripotency genes. We have also conducted ChIP-seq analysis for Pol II binding to demonstrate that SE-associated genes depend on BRD4-dependent Pol II binding at TSS and gene body for their productive transcriptional elongation. Overall design: Total RNA was extracted from samples using the RNeasy Qiagen kit according to the manufacturer’s instructions. Deep sequencing of RNA (1ug) from hESCs FGF- or MS436-treated at day 1 and day 5 was performed as described in (Higgin et al., 2010c). Samples were subjected to PolyA selection using magnetic oligo-dT beads. The resulting RNA samples were then used as input for library construction as described by the manufacturer (Illumina, CA, USA). RNA libraries were then sequenced on the GAIIx system using 50bp single reads. Chromatin for ChIP-sequencing was obtained from FGF-maintained hESCs, vehicle or MS417-treated (at 250nM concentration for 6h) (10 to 20x106 cells/IP). ChIP-Seq libraries were generated using standard Illumina kit and protocol as described in (Ntziachristos et al., 2012). We performed cluster amplification and single read 50 sequencing-method using the Illumina HiSeq 2000, following manufacturer’s protocols.
Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.
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Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma.
Cell line
View SamplesHere we report a novel role for H2A.Z.2 (H2AFV) as a mediator of cell proliferation and sensitivity to targeted therapies in malignant melanoma. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation of melanoma cells, which occurs via a G1/S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, and that such genes acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic melanoma cells. We further identified the BET family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and demonstrate that H2A.Z.2 silencing cooperates with BET inhibition to induce cell death.
Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma.
Cell line
View SamplesHere we report a novel role for H2A.Z.2 (H2AFV) as a mediator of cell proliferation and sensitivity to targeted therapies in malignant melanoma. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation of melanoma cells, which occurs via a G1/S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, and that such genes acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic melanoma cells. We further identified the BET family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and demonstrate that H2A.Z.2 silencing cooperates with BET inhibition to induce cell death. Overall design: Expression levels for non tumorigenic (Melanocytes) and human melanoma cell line SKmel147, before and after JQ1 treatement
Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma.
No sample metadata fields
View SamplesOne of the key aspects of neuronal differentiation is the array of neurotransmitters and neurotransmitter receptors that each neuron possesses. One important goal of developmental neuroscience is to understand how these differentiated properties are established during development. In this paper, we use fluorescence activated cell sorting and RNA-seq to determine the transcriptome of the Drosophila CNS midline cells, which consist of a small number of well-characterized neurons and glia. These data revealed that midline cells express 9 neuropeptide precursor genes, 13 neuropeptide receptor genes, and 31 small-molecule neurotransmitter receptor genes. In situ hybridization and high-resolution confocal analyses were carried-out to determine the midline cell identity for these neuropeptides and the neuropeptide receptors. The results revealed a surprising level of diversity. Neuropeptide genes are expressed in a variety of midline cell types, including motoneurons, GABAergic interneurons, and midline glia. These data revealed previously unknown functional differences among the highly-related iVUM neurons. There also exist segmental differences in expression for the same neuronal sub-type. Similar experiments on midline-expressed neuropeptide receptor genes reveal considerable diversity in synaptic inputs. Multiple receptor types were expressed in midline interneurons and motoneurons, and, in one case, link feeding behavior to gut peristalsis and locomotion. There were also segmental differences, variations between the 3 iVUMs, and three hormone receptor genes were broadly expressed in most midline cells. The Drosophila Castor transcription factor is present at high levels in iVUM5, which is both GABAergic and expresses the short neuropeptide F precursor gene. Genetic and misexpression experiments indicated that castor specifically controls expression of the short neuropeptide F precursor gene, but does not affect iVUM cell fate or expression of Gad1. This indicates a novel function for castor in regulating neuropeptide gene expression. Overall design: To study the development and differentiation of the CNS midline cells of Drosophila melanogaster on a genome-wide scale, these cells were labeled with GFP using the GAL/UAS system and FACS purified at 2 ermbryonic time-points; 6-8 hours and 14-16 hours after egg laying. Poly(A) mRNA was collected from these samples and cDNA libraries were generated. Sequencing was performed on 6 independent samples: Two FACS purified CNS-midline cell samples and one non-midline sample taken from 6-8 hours After Egg Laying (AEL) embryos and from 14-16 hours AEL embryos.
Transcriptome analysis of Drosophila CNS midline cells reveals diverse peptidergic properties and a role for castor in neuronal differentiation.
Specimen part, Subject, Time
View SamplesBackground: In human malaria, parasites of the genus Plasmodium elicit expansion of atypical memory B cells (atMBCs), which lack the classical markers CD21 and CD27. We have identified a putative population of analogous B cells in a murine model of infection with P. chabaudi, delineated by the marker FCRL5. We performed RNA-Seq on FCRL5+ and FCRL5- B cells sorted from infected mice, so as to characterize the transcriptional profile of these cells and permit comparison to atMBCs in humans. Results: FCRL5+ B cells were found to have distinct transcriptional profiles from FCRL5- B cells, with approximately 400 genes exhibiting significant differences between the two groups. Additionally, about 25% of these differentially expressed genes were also differentially expressed in human atMBCs versus classical MBCs, as previously described by Sullivan et al (PLoS Pathogens 2015). Conclusions: FCRL5+ class-switched B cells are a transcriptionally distinct subset arising in P. chabaudi infection, with transcriptional similarities to human atMBCs that develop in chronic malaria settings. Overall design: Class-switched B cells (IgM- IgD- CD19+) were isolated into FCRL5+ and FCRL5- populations by double-sorting from the blood of C57BL/6 adult female mice 21 days post-infection with Plasmodium chabaudi. Pools of ~1000 cells were isolated and processed for RNA sequencing. 5 biological replicates were analyzed for each sample type.
FCRL5<sup>+</sup> Memory B Cells Exhibit Robust Recall Responses.
Specimen part, Subject
View SamplesInhibitor of apoptosis (IAP) proteins constitute a conserved family of molecules which regulate both apoptosis and receptor signaling. They are often deregulated in cancer cells and represent potential targets for therapy. In our work, we investigated the effect of IAP inhibition in vivo to identify novel downstream genes expressed in an IAP-dependent manner that could contribute to cancer aggressiveness. To this end, immunocompromised mice engrafted subcutaneously with the triple negative breast cancer MDA-MB231 cell line were treated with SM83, a pan-IAP inhibitor developed by us, and tumor nodules were profiled for gene expression. Our work suggests that IAP-targeted therapy could contribute to EGFR inhibition and the reduction of its downstream mediators. This approach could be particularly effective in cells characterized by high levels of EGFR and Snai2, such as triple negative breast cancer.
cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells.
Specimen part, Treatment
View SamplesWe examined the role of TREM2 on microglia responses to demyelination
TREM2 sustains microglial expansion during aging and response to demyelination.
Sex, Specimen part, Time
View SamplesBackground: In malaria, parasites of the genus Plasmodium elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. In a microarray analysis of pooled, activated CD4+ T cells from mice infected with P. chabaudi, we detected inducible expression of Csf1, which promotes macrophage proliferation. To better characterize Csf1-producing T cells, single-cell RNA-Seq was performed. Results: Robust Csf1 expression was detected in a subset of sampled CD4+ T cells (n = 14/35), whereas the remainder of cells had no detectable Csf1. Further, we identified ~ 400 genes that were differentially expressed between Csf1+ and Csf1- T cells. Conclusions: This work defines the transcriptional landscape of a subset of activated CD4+ T cells that produce the cytokine Csf1. These cells are expected to be important in infections with intracellular pathogens such as Plasmodium. Overall design: Antigen-experienced (CD11a+ CD49d+) CD4+ T cells were isolated by double-sorting from the blood of C57BL/6 adult female mice 6 days post-infection with Plasmodium chabaudi. Single cells were isolated and processed for RNA sequencing using a Fluidigm C1 integrated fluidic circuit chip. 35 biological replicates were analyzed.
Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection.
Sex, Specimen part, Subject, Time
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