Project description:Translational profiling methodologies enable the systematic characterization of cell types in complex tissues such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy to profile CNS cell types in a spatiotemporally-restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by TRAP. We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting AAV injections enabled the elucidation of regional differences in gene expression within this cell type. Taken together, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre.
Project description:Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, Translating Ribosome Affinity Purification (TRAP), permits comprehensive studies of translated mRNAs in genetically defined cell populations following physiological perturbations. To establish the generality of this approach, we present translational profiles for twenty four CNS cell populations, and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole tissue microarray studies, and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of sixteen transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of CNS structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well known and previously uncharacterized neural cell populations. Keywords: Cell Type Comparison
Project description:Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, Translating Ribosome Affinity Purification (TRAP), permits comprehensive studies of translated mRNAs in genetically defined cell populations following physiological perturbations. To establish the generality of this approach, we present translational profiles for twenty four CNS cell populations, and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole tissue microarray studies, and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of sixteen transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of CNS structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well known and previously uncharacterized neural cell populations. Keywords: Cell Type Comparison For each cell population, three independent TRAP replicates were collected, and total RNA from both the immunoprecipitate and unbound fractions were seperately amplified and hybridized. For each tissue, several representative unbound fractions are provided to serve as controls. Biological replicates are GCRMA normalized within groups. Following averaging of replicates, we recommend further global normalization between groups, using affymetrix biotinylated controls, to correct for any broad biases in scanning and hybridization. Finally for many analyses, we also recommend filtering to remove those probesets with low IP/UB fold change values from each cell type. Researchers can contact us for spreadsheets where these additional steps have been completed.
Project description:Single-cell analysis has become a powerful approach for the molecular characterization of complex tissues. Methods for quantifying gene expression and chromatin accessibility2 of single cells are now well-established, but analysis of chromatin regions with specific histone modifications has been technically challenging. Here, we adapt the recently published CUT&Tag method3 to scalable single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues. We focus on profiling Polycomb Group (PcG) silenced regions marked by H3K27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we use scCUT&Tag to profile H3K27me3 in a brain tumor patient before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment.
Project description:Molecular profiling of human primary cell types is essential for holistic understanding of human biology. Here we present a map of 28 human primary cell types with comprehensive measurements of expressed transcripts and proteins. Three major clusters of epithelial, endothelial, and mesenchymal cell types were observed both at transcriptome and proteome level along with discovery of several cell type specific proteins. Among them, we characterized the novel epithelial specific protein C1orf116 which was further validated with immunohistochemistry across various human tissues. Comprehensive proteome profiling across cell types allowed characterization of ten orphan proteins including TMEM252 and TM4SF18 which lacked protein level evidence. Exhaustive protein database search considering 27 biologically relevant post-translational modifications and 12 chemical modifications was performed, thanks to the increased performance of cloud-based search engine. This, for the first time, provided the catalog of post-translational modifications across cell types, which serve as a resource of PTMs for future studies. We characterized proteins such as protein EEF1A1with various types of PTMs and understudied PTMs including histidine methylation, and O-acetylation of serine. Interestingly, the unexpected higher frequency of dioxidation on tryptophan than that of methionine led us to characterize oxidative mitochondria complex subunit proteins modified through oxidative phosphorylation in mitochondria, which was mostly considered as chemical artifact so far. Overall, these indicated that considering various PTMs during protein database search from non-enriched samples could be routinely used in proteomics experiments. Further, protein database search strategy considering alternative translational start sites, splice junctions, and translational readthrough provided a unique opportunity to refine genome annotation with their proteomic evidence. We believe that our data with the large catalog of transcriptome and proteome of normal human primary cells provide a cornerstone for holistic understanding of cell biology.
Project description:Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), were missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we used Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis revealed genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells (MDCs). Importantly, we detected heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observed remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.
2020-11-21 | GSE160048 | GEO
Project description:Molecular profiling identifies at least 3 distinct types of post-transplant lymphoproliferative disorder involving CNS
Project description:Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), were missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we used Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis revealed genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells (MDCs). Importantly, we detected heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observed remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.