Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1177590 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Systematic reconstruction of molecular cascades regulating growth plate development using single-cell RNA-seq
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Project description:With the recent advancements in genome editing, next generation sequencing (NGS), and scalable cloning techniques, scientists can now conduct genetic screens at unprecedented levels of scale and precision. With such a multitude of technologies, there is a need for a simple yet comprehensive pipeline to enable systematic mammalian genetic screening. In this study, we develop novel algorithms for target identi fication and a toxin-less Gateway cloning tool, termed MegaGate, for library cloning which, when combined with existing genetic perturbation methods and NGS-coupled readouts, enable versatile engineering of relevant mammalian cell lines. Our integrated pipeline for Sequencing-based Target Ascertainment and Modular Perturbation Screening (STAMPScreen) can thus be utilized for a host of cell state engineering applications.
2021-08-14 | GSE182088 | GEO
Project description:Recent advancements in functional genomics have provided an unprecedented ability to measure diverse molecular modalities, but learning causal regulatory relationships from observational data remains challenging. Here, we leverage pooled genetic screens and single cell sequencing (i.e. Perturb-seq) to systematically identify the targets of signaling regulators in diverse biological contexts. We demonstrate how Perturb-seq is compatible with recent and commercially available advances in combinatorial indexing and next-generation sequencing, and perform more than 1,500 perturbations split across six cell lines and five biological signaling contexts. We introduce an improved computational framework (Mixscale) to address cellular variation in perturbation efficiency, alongside optimized statistical methods to learn differentially expressed gene lists and conserved molecular signatures. Finally, we demonstrate how our Perturb-seq derived gene lists can be used to precisely infer changes in signaling pathway activation for in-vivo and in-situ samples. Our work enhances our understanding of signaling regulators and their targets, and lays a computational framework towards the data-driven inference of an ‘atlas’ of perturbation signatures.
2024-11-04 | GSE281048 | GEO
Project description:Scalable, compressed phenotypic screening using pooled perturbations (PDAC screens)
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Project description:Perturb-seq: Dissecting molecular circuits with scalable single cell RNA profiling of pooled genetic screens
| PRJNA354362 | ENA
Project description:The growth plate, which comprises sequentially differentiated cell layers, is a critical structure for bone elongation and regeneration. Although several key regulators in growth plate development have been identified using primarily genetic perturbation, the systematic understanding is still limited. Here we used single cell RNA-seq to interrogate gene expression profiles of 217 single cells from growth plates, and developed the bioinfromatics pipeline Sinova to de-novo reconstruct physiological growth plate development in both temporal and spatial high-resolution. Our unsupervised model not only confirmed prior knowledge but also enabled systematic discovery of novel genes, potential signal pathways and surface markers CD9/CD200 to precisely depict the development. Sinova further identified effective transcriptional factor portfolio directing growth plate maturation, which was cross-validated experimentally using an in-vitro EGFP-Col10a screening system. Our case demonstrated systematic reconstructing of molecular cascades of a developmental process from single-cell profiling, and the workflow is readily transferable to other physiological scenarios. 217 single-cell RNA-seq for cell isolated from mouse growth plate at postnatal day7
2016-06-14 | E-GEOD-76157 | biostudies-arrayexpress
Project description:Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow rich phenotyping through single-cell RNA sequencing readouts, and separately, characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
2023-05-02 | GSE215253 | GEO
Project description:Genetic screens using CRISPR technology are widely exploited to explore novel targets and discover resistance mechanisms of cancer treatments, including cancer immunotherapy. As it is a time- and resource-intensive manner to individually validate potential candidates and clarify their working mechanisms, single-cell CRISPR screen platforms are designed and developed to parallelly characterize these factors and explore underlying mechanisms in a high-throughput manner. By leveraging the resources generated in our previous studies, Perturb-seq featured with capture sequences integrated into gRNA-expressing vectors and CROP-seq featured with poly(A)-tailed gRNA transcripts under the control of RNAPII promoters were selected among several independent single-cell CRISPR screen platforms to directly evaluate the performance of different scCRISPR screen platforms in determining underlying mechanisms of tumor intrinsic immune regulation and optimize experimental conditions and data analysis in immune-related scCRISPR screens. In the study, the influence of each perturbation on the gene expression profile and the response to in vitro T cell killing and in vivo anti-PD-1 treatment were comprehensively appraised at the single-cell level. It is clearly demonstrated that scCRISPR screen platforms could efficiently characterize gene expression profiles and perturbation effects present in individual cells during both in vitro and in vivo immune-related screen processes. Particularly, scCRISPR screen platforms presented more detail to determine heterogeneous change of gene expression in distinct knockout tumor cells in the presence of immune attack which cannot be easily assessed by canonical bulk RNA-seq. Collectively, scCRISPR screen technologies provide scalable and reliable platforms to validate identified tumor intrinsic immune factors identified from genome-wide CRISPR screens and elucidate their potential working mechanisms.
2022-11-13 | GSE206107 | GEO
Project description:The expression of inhibitory immune checkpoint molecules such as PD-L1 is frequently observed in human cancers and can lead to the suppression of T-cell mediated immune responses. Here we apply ECCITE-seq, a technology which combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1, and leverage our multi-modal data to identify both transcriptional and post-transcriptional modes of regulation. In particular, we discover that the kelch-like protein KEAP1 and the transcriptional activator NRF2, mediate levels of PD-L1 upregulation after IFNγ stimulation. Our results identify a novel mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multi-modal single-cell perturbation screens.
2020-06-29 | GSE153056 | GEO