Targeted single-cell transcriptome readouts for high-throughput genetics and functional genomics
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA559094 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10885102_1.fastq.gz | Fastqsanger.gz | |||
| SRR10885102_2.fastq.gz | Fastqsanger.gz | |||
| SRR10885103_1.fastq.gz | Fastqsanger.gz | |||
| SRR10885103_2.fastq.gz | Fastqsanger.gz | |||
| SRR10885104_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 238 |
Similar Datasets
Project description:We show that targeted single-cell RNA-sequencing (TAP-seq) permits reliable mapping of cell (sub)types with as little as 100 reads per cell and reduces the sequencing costs for differential expression testing by a factor of 10-30, thereby enabling a cost-effective profiling of a large number of genotypes at the single cell level. We demonstrate the use of TAP-seq by generating comprehensive perturbation-based enhancer-target gene maps for 1.5% of the human genome.
2020-03-23 | GSE135497 | GEO
Project description:Identification of CD8+ T cell epitopes is critical for the development of immunotherapeutics. Existing methods for major histocompatibility complex class I (MHC class I) ligand discovery are time intensive, specialized and unable to interrogate specific proteins on a large scale. Here, we present EpiScan, which uses surface MHC class I levels as a readout for whether a genetically encoded peptide is an MHC class I ligand. Predetermined starting pools composed of >100,000 peptides can be designed using oligonucleotide synthesis, permitting large-scale MHC class I screening. We exploit this programmability of EpiScan to uncover an unappreciated role for cysteine that increases the number of predicted ligands by 9-21%, reveal affinity hierarchies by analysis of biased anchor peptide libraries and screen viral proteomes for MHC class I ligands. Using these data, we generate and iteratively refine peptide binding predictions to create EpiScan Predictor. EpiScan Predictor performs comparably to other state-of-the-art MHC class I peptide binding prediction algorithms without suffering from underrepresentation of cysteine-containing peptides. Thus, targeted immunopeptidomics using EpiScan will accelerate CD8+ T cell epitope discovery toward the goal of individual-specific immunotherapeutics.
| S-EPMC10314971 | biostudies-literature
Project description:The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.
| S-EPMC5630457 | biostudies-literature
Project description:Substantial intrastrain variation at the nucleotide level complicates molecular and genetic studies in zebrafish, such as the use of CRISPRs or morpholinos to inactivate genes. In the absence of robust inbred zebrafish lines, we generated NHGRI-1, a healthy and fecund strain derived from founder parents we sequenced to a depth of ∼50×. Within this strain, we have identified the majority of the genome that matches the reference sequence and documented most of the variants. This strain has utility for many reasons, but in particular it will be useful for any researcher who needs to know the exact sequence (with all variants) of a particular genomic region or who wants to be able to robustly map sequences back to a genome with all possible variants defined.
| S-EPMC4174928 | biostudies-literature
Project description:The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.
| S-EPMC4813485 | biostudies-other
Project description:Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 °C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.
| S-EPMC3345369 | biostudies-literature
Project description:Materials genomics represents a research mode for materials development, for which reliable methods for efficient materials construction are essential. Here we present a methodology for high-throughput construction of covalent organic frameworks (COFs) based on materials genomics strategy, in which a gene partition method of genetic structural units (GSUs) with reactive sites and quasi-reactive assembly algorithms (QReaxAA) for structure generation were proposed by mimicking the natural growth processes of COFs, leading to a library of 130 GSUs and a database of ~470,000 materials containing structures with 10 unreported topologies as well as the existing COFs. As a proof-of-concept example, two generated 3D-COFs with ffc topology and two 2D-COFs with existing topologies were successfully synthesized. This work not only presents useful genomics methods for developing COFs and largely extended the COF structures, but also will stimulate the switch of materials development mode from trial-and-error to theoretical prediction-experimental validation.
| S-EPMC6288119 | biostudies-literature
Project description:BackgroundThe ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.ResultsAiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.ConclusionsUsing this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.
| S-EPMC3260098 | biostudies-literature
Project description:SummaryPreparing functional genomic (FG) data with diverse assay types and file formats for integration into analysis workflows that interpret genome-wide association and other studies is a significant and time-consuming challenge. Here we introduce hipFG (Harmonization and Integration Pipeline for Functional Genomics), an automatically customized pipeline for efficient and scalable normalization of heterogenous FG data collections into standardized, indexed, rapidly searchable analysis-ready datasets while accounting for FG datatypes (e.g. chromatin interactions, genomic intervals, quantitative trait loci).Availability and implementationhipFG is freely available at https://bitbucket.org/wanglab-upenn/hipFG. A Docker container is available at https://hub.docker.com/r/wanglab/hipfg.
| S-EPMC10660288 | biostudies-literature
Project description:Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts functional readouts.
| PRJNA911760 | ENA