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Homo sapiens

ABSTRACT: Polysome-profiling in small tissue samples - proof of concept experiment for the use of an optimized non linear gradient

OTHER RELATED OMICS DATASETS IN: E-MTAB-9628

PROVIDER: PRJNA390060 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5666630.fastq.gz	Fastqsanger.gz
	SRR5666640.fastq.gz	Fastqsanger.gz
	SRR5666650.fastq.gz	Fastqsanger.gz
	SRR5666631.fastq.gz	Fastqsanger.gz
	SRR5666641.fastq.gz	Fastqsanger.gz

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Polysome-profiling in small tissue samples - proof of concept experiment for the use of an optimized non linear gradient

Project description:Polysome-profiling is commonly used to study translatomes, i.e. transcriptome-wide patterns of translational efficiency. The standard approach for collecting efficiently translated polysome-associated RNA results in laborious extraction of RNA from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts such as primary cells or frozen tissues. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for mRNAs associated with >3 ribosomes in only one or two fractions, thereby reducing sample handling 5-10 fold. The technique can be applied to frozen tissue samples from biobanks, and generates polysome-associated RNA with a quality reflecting the starting material. When coupled with smart-seq2, a single-cell RNA sequencing technique, translatomes from small tissue samples can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associated with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation to maintain protein levels constant. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in bio banks.

2017-10-03 | GSE99909 | GEO

Project description:Enspyre Proof-of-Concept

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Polysome-seq in zebrafish early embryonic development

Project description:We followed the polysomal association of maternal and early zygotic transcriptome over the first few hours of embryonic development, prior to and after MBT. We isolated polysome-associated (bound) and non-polysome-associated (unbound) mRNAs using sucrose gradient centrifugation followed by size fractionation. Using next generation sequencing (RNA-seq), we profiled the transcriptome in polysome-bound and unbound fractions. Our analysis revealed distinct dynamics of polysome association of cytoplasmically polyadenylated maternal mRNAs.

2017-10-14 | GSE88776 | GEO

Genome-wide analysis of host mRNA translation during hepatitis C virus infection

Project description:Sucrose gradient ultracentrifugation followed by microarray analysis was used to identify translationally-regulated mRNAs (mRNAs associated with ribosomes) from JFH1-infected and uninfected Huh-7.5.1 cells. Huh7 cells were infected with HCV JFH-1 strain. Controls consisted in non-infected cells and infected cells treated with an anti-protease. 3 days after infection, cell lysates were subjected to sucrose-gradient centrifugation in order to isolate polysome-free RNAs from polysome-bound (translated) RNAs. The non-polysomal, polysomal as well as cytosolic (non fractionned) RNAs, from infected, non-infected, and infected-treated cells were subject sur microarray analysis.

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Profiling of free and polysome-bound mRNA from exponential and stationary growth phase cultures of Haloferax volcanii to identify growth phase dependent translational regulation

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Project description:In order to detect changes in liver tissue protein translation efficiency during liver regeneration, we performed sucrose density gradient centrifugation to separate translated (associated with polysome fraction), and non-translated (associated with sub-polysome fraction) transcripts in extracts from mouse livers 0 h and 24 h post-surgery. We found that compared to liver tissues before surgery, extracts from tissues 24 h post-surgery had significantly fewer transcripts associated with polysomes, suggesting a reduction in protein synthesis activity.

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