Next Generation Sequencing Facilitates Quantitative Analysis of mock oligodendrocytes (Ols) and tdTomato+ and tdTomato- Ols Transcriptomes.
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ABSTRACT:
PROVIDER: PRJNA625280 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11544527.fastq.gz | Fastqsanger.gz | |||
| SRR11544528.fastq.gz | Fastqsanger.gz | |||
| SRR11544529.fastq.gz | Fastqsanger.gz | |||
| SRR11544530.fastq.gz | Fastqsanger.gz | |||
| SRR11544531.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: Comparision transcriptomes of isolated tdTomato+ and tdTomato- OLs from the brains and spinal cords of infected mice at 30 dpi to those of OLs isolated from mock-infected central nervous system (CNS) samples by nextgen sequencing Methods: Brains and spinal cords were harvested at day 30 p.i. and processed as described above. After Live/Dead staining and treatment with Fc block, OLs were identified using APC-anti-O4 (1:50; Miltenyi). RNA was extracted from sorted OLs using an RNeasy Micro Kit (Qiagen). RNA sequence analysis was performed at the University of Minnesota Genomics Center. Results: We identified 158 and 115 genes, that showed a significant difference (p<0.05) when tdTomato+ and tdTomato- cells were compared to cells from mock-infected mice, respectively, with 61 mRNAs common to both tdTomato- and tdTomato+ cells
2020-04-15 | GSE148650 | GEO
Project description:microRNA expression profiles in two stage-specific populations of oligodendrocytes: OPCs expressing the A2B5 ganglioside (A2B5+ cells) and OLs that are positive for the galactocerebroside marker (GalC+ cells). Cells were isolated postnatal day 7.
2008-11-11 | E-MEXP-1589 | biostudies-arrayexpress
Project description:In the central nervous system (CNS), myelin formation by oligodendrocytes (OLs) relies on actin dynamics. Actin polymerization supports the ensheathment step, when the OL process contacts and surrounds a selected portion of axon. While a drastic shift to actin depolymerization is then required to enable the wrapping process and the formation of a multilayered myelin sheath. Molecular mechanisms regulating this switch to actin depolymerization are not fully elucidated. P21-activated kinase 1 (PAK1), a downstream effector of Rho GTPases Rac1 and Cdc42, highly expressed in OLs, can regulate actin dynamics through its kinase activity. We found that PAK1 loss-of-function in OLs, in vivo, stimulates the wrapping and the thickening of myelin sheaths. We showed that PAK1 is highly expressed in myelinating OLs, yet in its inactive form, the one with the ability to trigger actin disassembly. Interestingly, we found that modulations of PAK1 kinase activity control actin dynamics in OLs, thereby myelin formation. Our data demonstrate that PAK1 inactivation in OLs is required for actin depolymerization and proper myelin formation, suggesting the presence of an endogenous PAK1 inhibitor in myelinating OLs. Proteomic analysis of PAK1 binding partners enabled us to identify several proteins likely to regulate its activity.
2024-06-03 | PXD044344 | Pride
Project description:A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate astrocytes, neurons, and oligodendrocytes. Here we describe the first method for the isolation and purification of developing and mature astrocytes from mouse forebrain. This method takes advantage of the expression of S100β by astrocytes. We used fluorescent activated cell sorting (FACS) to isolate EGFP positive cells from transgenic mice that express EGFP under the control of an S100β promoter. By depletion of astrocytes and oligodendrocytes we obtained purified populations of neurons, while by panning with oligodendrocyte-specific antibodies we obtained purified populations of oligodendrocytes. Using GeneChip Arrays we then created a transcriptome database of the expression levels of over 20,000 genes by gene profiling these three main CNS neural cell types at postnatal ages day 1 to 30. This database provides the first global characterization of the genes expressed by mammalian astrocytes in vivo and is the first direct comparison between the astrocyte, neuron, and oligodendrocyte transcriptomes. We demonstrate that Aldh1L1, a highly expressed astrocyte gene, is a highly specific antigenic marker for astrocytes with a substantially broader, and therefore potentially more useful, pattern of astrocyte expression than the traditional astrocyte marker GFAP. This transcriptome database of acutely isolated and highly pure populations of astrocytes, neurons and oligodendrocytes provides a resource to the neuroscience community by providing improved cell type specific markers and for better understanding of neural development, function, and disease. We acutely purified mouse astrocytes from early postnatal ages (P1) to later postnatal ages (P30), when astrocyte differentiation is morphologically complete (Bushong et al., 2004), and acutely purified mouse OL-lineage cells from stages ranging from OPCs to newly differentiated OLs to myelinating OLs. We extracted RNA from each of these highly purified, acutely isolated cell types and used GeneChip Arrays to determine the expression levels of over 20,000 genes and construct a comprehensive database of cell type specific gene expression in the mouse forebrain. Analysis of this database confirms cell type specific expression of many well characterized and functionally important genes. In addition, we have identified thousands of new cell type enriched genes, thereby providing important new information about astrocyte, OL, and neuron interactions, metabolism, development, and function. This database provides a comparison of the genome-wide transcriptional profiles of the main CNS cell types and is a resource to the neuroscience community for better understanding the development, physiology, and pathology of the CNS. Keywords: Developmental CNS Cell type comparision FACS purification of astrocytes: Dissociated forebrains from S100β-EGFP mice were resuspended in panning buffer (DBPS containing 0.02% BSA and 12.5 U/ml DNase) and sequentially incubated on the following panning plates: secondary antibody only plate to deplete microglia, O4 plate to deplete OLs, PDGFRα plate to deplete OPCs, and a second O4 plate to deplete any remaining OLs. This procedure was sufficient to deplete all OL-lineage cells from animals P8 and younger, however, in older animals that had begun to myelinate, additional depletion of OLs and myelin debris was accomplished as follows. The nonadherent cells from the last O4 dish were harvested by centrifugation, and the cells were resuspended in panning buffer containing GalC, MOG, and O1 supernatant and incubated for 15 minutes at room temperature. The cell suspension was washed and then resuspended in panning buffer containing 20 μg donkey anti-mouse APC for 15 minutes. The cells were washed and resuspended in panning buffer containing propidium iodide (PI). EGFP+ astrocytes were then purified by fluorescence activated cell sorting (FACS). Dead cells were gated out using high PI staining and forward light scatter. Astrocytes were identified based on high EGFP fluorescence and negative APC fluorescence from indirect immunostaining for OL markers GalC, MOG, and O1. Cells were sorted twice and routinely yielded >99.5% purity based on reanalysis of double sorted cells.; FACS purification of neurons: EGFP- cells were the remaining forebrain cells after microglia, OLs, and astrocytes had been removed, and were primarily composed of neurons, and to a lesser extent, endothelial cells (we estimate < 4% endothelial cells at P7 and < 20% endothelial cells at P17). EGFP- cells from S100β-EGFP dissociated forebrain were FACS purified in parallel with astrocyte purification and were sorted based on their negative EGFP fluorescence immunofluorescence. Cells were sorted twice and routinely yielded >99.9% purity. In independent preparations, the EGFP- cell population was additionally depleted of endothelial cells and pericytes by sequentially labeling with biotin-BSL1 lectin and streptavidin-APC while also labeling for OL markers as described above. Cells were sorted twice and routinely yielded >99.9% purity.; Panning purification of oligodendrocyte lineage cells: Dissociated mouse forebrains were resuspended in panning buffer. In order to deplete microglia, the single-cell suspension was sequentially panned on four BSL1 panning plates. The cell suspension was then sequentially incubated on two PDGFRα plates (to purify and deplete OPCs), one A2B5 plate (to deplete any remaining OPCs), two MOG plates (to purify and deplete myelinating OLs), and one GalC plate (to purify the remaining PDGFRα-, MOG-, OLs). The adherent cells on the first PDGFRα, MOG, and GalC plates were washed to remove all antigen-negative nonadherent cells. The cells were then lysed while still attached to the panning plate with Qiagen RLT lysis buffer, and total RNA was purified. Purified OPCs were >95% NG2 positive and 0% MOG positive. Purified Myelin OLs were 100% MOG positive, >95% MBP positive, and 0% NG2 positive. Purified GalC OLs depleted of OPCs and Myelin OLs were <10% MOG positive and ~50% weakly NG2 positive, a reflection of their recent development as early OLs.; Data normalization and analysis: Raw image files were processed using Affymetrix GCOS and the MAS 5.0 algorithm. Intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and absent/present calls for individual probe sets were determined. Gene expression values were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model. (www.dchip.org, Li and Wong, 2001). The 29 samples were grouped into 9 sample types: Astros P7-P8, Astros P17, Astros P17-gray matter (P17g), Neurons P7, Neurons P17, Neurons-endothelial cell depleted (P7n, P17n), OPCs, GalC-OLs, and MOG-OLs. Gene filtering was performed to select probe sets that were consistently expressed in at least one cell type, where consistently expressed was defined as being called present and having a MAS 5.0 intensity level greater than 200 in at least two-thirds of the samples in the cell type. We identified 20,932 of the 45,037 probe sets that were consistently expressed in at least one of the nine cell types. The Significance Analysis of Microarrays (SAM) method (Tusher et al., 2001) was used to determine genes that were significantly differentially expressed between different cell types (see Supplemental Table S2 for SAM cell type groupings). Clustering was performed using the hclust method with complete linkage in R. Expression values were transformed for clustering by computing a mean expression value for the gene using those samples in the corresponding SAM statistical analysis, and then subtracting the mean from expression intensities. In order to preserve the log2 scale of the data, unless otherwise indicated, no normalization by variance was performed. Plots were created using the gplots package in R. The Bioconductor software package (Gentleman et al., 2004) was used throughout the expression analyses. Functional analyses were performed through the use of Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).
2007-12-30 | E-GEOD-9566 | biostudies-arrayexpress
Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Mock/MASCp36 infected mouse lung Transcriptomes
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2022-08-26 | GSE211739 | GEO
Project description:Oligodendrocytes (OLs) are glial cells within the central nervous system (CNS) that produce myelin, which coats neuronal axons and enhances fast propagation of neuronal signals. OLs are known to be severely affected by alcohol in fetal alcohol spectrum disorder (FASD) and alcohol use disorder (AUD). In FASD, oligodendrocyte precursor cells have been reported to be highly affected by any dose of alcohol. In AUD, high doses of alcohol can lead to demyelinating complications such as Wernicke-Korsakoff sybdrome. However, relatively little is known about how alcohol at low and high doses affects mature OLs (mOLs). To better understand the impact of concentration-dependent alcohol exposure on mOLs, we isolated and differentiated immature murine OLs into mOLs and exposed them to a moderate or high concentration of alcohol in vitro. RNA was then isolated from the cultures and sequenced using 3’-Tag RNA sequencing. Differential expression across alcohol concentration and control groups was calculated, which was used for gene set enrichment analysis to identify KEGG and GO terms that were significantly changed as a result of alcohol treatment. We found concentration dependent changes in biological pathways related to cell cycle regulation, translation initiation, integrin signaling, FGF2 signaling, inflammation, and myelination. Further, we found that the moderate and high concentrations of alcohol elicited distinct changes in gene expression, indicating that concentration is a critical factor when determining the effects of alcohol on transcriptomic changes.
2023-09-24 | GSE239691 | GEO
Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Mock/ZIKV infected Guinea Pig Lung Transcriptomes
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Project description:Multiple sclerosis (MS) is characterized by the loss of myelin and of myelin-producing oligodendrocytes (OLs) in the central nervous system (CNS). Pro-inflammatory CD4+ Th17 cells are considered pathogenic in MS and are harmful to OLs. We investigated the mechanisms driving human CD4+ T cell-mediated OL cell death. Using single cell RNA sequencing, we assessed the transcriptomic profile of primary human OLs and Th17 cells in direct contact or separated by an insert. We showed that upon close interaction, OLs upregulate the expression of mRNA coding for chemokines and antioxidant/anti-apoptotic molecules, while Th17 cells upregulate the expression of mRNA coding for chemokines and pro-inflammatory cytokines such as IL-17A, IFN-γ and granzyme B. We found that secretion of CCL3, CXCL10, IFN-γ, TNFα and granzyme B is induced upon direct contact in co-cultures of human Th17 cells with human OLs. In addition, we validated by flow cytometry and immunofluorescence that granzyme B levels are upregulated in Th17 compared to Th2 cells, and are even higher in Th17 cells upon direct contact with OLs or MO3.13 cells compared to Th17 cells separated from OLs by an insert.
2022-02-28 | GSE196953 | GEO