Ontology highlight
ABSTRACT:
PROVIDER: PRJEB31612 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Recently, RNA sequencing has achieved single cell resolution, but what is limiting is an effective way to routinely isolate and process large numbers of individual cells for in-depth sequencing, and to do so quantitatively. We have developed a droplet-microfluidic approach for parallel barcoding thousands of individual cells for subsequent RNA profiling by next-generation sequencing. This high-throughput method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. Using this technique, we analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility and low noise of this high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships.
2015-05-20 | GSE65525 | GEO
Project description:Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate
cell subpopulations with a high level of recovery and accuracy. However, the cell sorting
procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and a microfluidic chip-based sorting approach on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with higher transcriptional and spliceosomal regulation and mechanical stress signaling. These results
indicate microfluidic chip-based sorting is less disruptive compared to droplet-based sorting.
2023-11-09 | MSV000093348 | MassIVE
Project description:To expedite immunotherapy development, better analysis of treatment efficacy at early in vitro stages is needed. Using a droplet-based microfluidic platform, we have established a method for multi-parameter quantifiable phenotypic and genomic observations of immunotherapies. Chimeric antigen receptor (CAR) NK cells provide treatment of interest in the current immunotherapy landscape and provide an optimal model for evaluating our novel methodology. For this approach, NK cells transduced with a CD19 CAR were compared to non-transduced NK cells in their ability to kill a lymphoma cell line. Using our novel single-cell droplet array platform, we quantified the increase in cytotoxicity and synaptic contact formation of CAR-NK cells over non-transduced NK cells. With our droplet sorter, we separated NK cells based on target cell killing for transcriptomic sequencing. Our data revealed expected improvement in cytotoxicity with the CD19 CAR but more importantly, provided unique insights into the factors involved in the cytotoxic mechanisms of CAR NK cells. This demonstrates a novel, improved system for immunotherapy screening.
2023-09-26 | GSE243787 | GEO
Project description:Recently, RNA sequencing has achieved single cell resolution, but what is limiting is an effective way to routinely isolate and process large numbers of individual cells for in-depth sequencing, and to do so quantitatively. We have developed a droplet-microfluidic approach for parallel barcoding thousands of individual cells for subsequent RNA profiling by next-generation sequencing. This high-throughput method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. Using this technique, we analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility and low noise of this high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships. A total of 8 single cell data sets are submitted: 3 for mouse embryonic stem (ES) cells (1 biological replicate, 2 technical replicates); 3 samples following LIF withdrawal (days 2,4, 7); one pure RNA data set (from human lymphoblast K562 cells); and one sample of single K562 cells.
2015-05-20 | E-GEOD-65525 | biostudies-arrayexpress
Project description:Droplet microfluidic methods have massively increased the throughput of single-cell RNA sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples as well as lower overall RNA capture efficiency. These drawbacks stem from the lack of strategies to enrich for high-quality material at the moment of cell encapsulation and the lack of implementable multi-step enzymatic processes that increase RNA capture. Here we alleviate both bottlenecks by deploying fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei or fixed cells and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half, depending on sample quality. We harness these unique properties to deliver a high-quality molecular atlas of mouse brain development using highly damaged input material. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.
2023-07-02 | GSE208156 | GEO
Project description:Single-cell RNA-seq of of dermal fibroblasts using microfluidic droplet capture (unstimulated cells)
| PRJEB22075 | ENA
Project description:Single-cell RNA-seq of of dermal fibroblasts using microfluidic droplet capture (stimulated cells)
| PRJEB22073 | ENA
Project description:High-throughput screening of neutralizing epitopes on HIV particles using single-virus droplet microfluidics
| PRJEB19490 | ENA
Project description:SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.
2023-03-10 | GSE200457 | GEO
Project description:SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.
2023-11-23 | GSE218732 | GEO