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Related Concept Videos

Ribosome Profiling02:24

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
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TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos
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Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification

Xicotencatl Gracida1, John A Calarco2

  • 1FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, United States; Department of Organismal and Evolutionary Biology, Harvard University, Cambridge, MA 02138, United States.

Methods (San Diego, Calif.)
|June 27, 2017
PubMed
Summary
This summary is machine-generated.

Translating Ribosome Affinity Purification (TRAP) enables cell-type specific transcriptome profiling in C. elegans. This method enriches translating mRNAs for genome-wide expression analysis, aiding discovery in response to stimuli.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Developmental Biology

Background:

  • Multicellular organisms rely on specialized cell types with unique gene expression patterns.
  • Profiling cell-type specific transcriptomes is crucial for understanding cellular specialization.
  • Existing methods for transcriptome profiling face challenges in invasiveness and accessibility.

Purpose of the Study:

  • To introduce and validate the Translating Ribosome Affinity Purification (TRAP) method for cell-type specific transcriptome profiling in C. elegans.
  • To provide a less invasive and powerful method for analyzing gene expression in defined cell types.
  • To leverage TRAP for discovering molecular responses to external or genetic perturbations.

Main Methods:

  • Utilized the TRAP technique in C. elegans, fusing a ribosomal protein to GFP under cell type-specific promoters.
  • Performed affinity purification of tagged ribosomes from animal lysates to enrich for cell-type specific translating mRNAs.
  • Generated cDNA libraries from purified mRNAs for high-throughput sequencing to obtain genome-wide transcript profiles.

Main Results:

  • Successfully adapted TRAP for C. elegans, enabling the isolation of translating mRNAs from specific cell types.
  • Demonstrated the utility of TRAP for genome-wide transcriptome profiling of genetically defined cell populations.
  • Established TRAP as a feasible method for studying gene expression changes in response to various stimuli.

Conclusions:

  • TRAP offers a powerful and accessible approach for cell-type specific transcriptome analysis in C. elegans.
  • The method facilitates the discovery of molecular components involved in cellular responses to perturbations.
  • TRAP enhances the study of gene expression dynamics in specific cell types, advancing our understanding of cellular specialization.