Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An Improved Platform for Functional Assessment of Large Protein Libraries in Mammalian Cells

ABSTRACT: An Improved Platform for Functional Assessment of Large Protein Libraries in Mammalian Cells

PROVIDER: PRJNA559843 | ENA |

REPOSITORIES: ENA

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	SRR9951730_1.fastq.gz	Fastqsanger.gz
	SRR9951730_2.fastq.gz	Fastqsanger.gz
	SRR9951740_1.fastq.gz	Fastqsanger.gz
	SRR9951740_2.fastq.gz	Fastqsanger.gz
	SRR9951750_1.fastq.gz	Fastqsanger.gz

Items per page:

1 - 5 of 146

Similar Datasets

An Improved Platform for Functional Assessment of Large Protein Libraries in Mammalian Cells

Project description:Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed, and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Finally, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.

2019-09-30 | GSE135717 | GEO

An improved platform for functional assessment of large protein libraries in mammalian cells.

Project description:Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.

| S-EPMC7145622 | biostudies-literature

A platform for functional assessment of large variant libraries in mammalian cells.

Project description:Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created. Second, a single variant from a library of transfected, promoter-less plasmids is recombined into the landing pad in each cell. Thus, every cell in the recombined pool expresses a single variant, allowing for parallel, sequencing-based assessment of variant effect. We describe a method for incorporating a single landing pad into a defined site of a cell line of interest, and show that our approach can be used generate more than 20 000 recombinant cells in a single experiment. Finally, we use our platform in combination with a sequencing-based assay to explore the N-end rule by simultaneously measuring the effects of all possible N-terminal amino acids on protein expression.

| S-EPMC5499817 | biostudies-literature

sequencing of yeast RNAi libraries for improved protein production

Project description:sequencing of yeast RNAi libraries for improved protein production

| PRJEB27502 | ENA

A safety screening platform for individualized cardiotoxicity assessment

Project description:A safety screening platform for individualized cardiotoxicity assessment

| PRJNA971626 | ENA

Improved characterization of single-cell RNA-seq libraries with paired-end avidity sequencing

Project description:Improved characterization of single-cell RNA-seq libraries with paired-end avidity sequencing

| PRJNA1140622 | ENA

Improved sensitivity of immunopeptidomics by leveraging available large-scale pan-HLA spectral libraries and data-independent acquisition

Project description:Mass spectrometry is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across HLA allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery-driven and data-dependent tandem mass spectrometry acquisition is commonly used, as it generates high quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that leads to lower sensitivity and reproducibility. In contrast, in data-independent acquisition, the fragmentation and acquisition of all fragment ions from a predefined list of precursor isolation windows yields a comprehensive map for a given sample. Because often the amount of HLA peptides eluted from biological samples, is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity - from sample-specific libraries to libraries combining from two to forty different immunopeptidomics samples. Matching DIA immunopeptidomics data against complex pan-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. We concluded that a comprehensive pan-HLA library for DIA approach is highly advantageous for the clinical Immunopeptidomics where low amount of biological sample is available for immunopeptidomics.

2021-04-13 | PXD022950 | Pride

An automatec microwell platform for large-scale single cell RNA-seq.

Project description:We report an automated microwell array platform for single cell RNA-seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.

2016-08-13 | GSE85575 | GEO

An improved method of constructing degradome library suitable for Illumina high-throughput sequencing platform

Project description:An improved method of constructing degradome library suitable for Illumina high-throughput sequencing platform

| PRJNA576350 | ENA

Small RNA libraries

Project description:Small RNA libraries

| PRJNA478199 | ENA