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Methods. 2017 Aug 15;126:130-137. doi: 10.1016/j.ymeth.2017.06.023. Epub 2017 Jun 23.

Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique.

Author information

1
FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, United States; Department of Organismal and Evolutionary Biology, Harvard University, Cambridge, MA 02138, United States. Electronic address: xgracida@fas.harvard.edu.
2
FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, United States; Department of Cell and Systems Biology, University of Toronto, Toronto M5S 3G5, Canada. Electronic address: john.calarco@utoronto.ca.

Abstract

Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods. Here, we describe the use of the Translating Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression patterns at the level of translating mRNAs. In TRAP, a ribosomal protein is fused to a tag (GFP) and is expressed under cell type-specific promoters to mark genetically defined cell types in vivo. Affinity purification of lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNAs are used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide profiles of transcripts from the targeted cell type. The ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to enable the discovery of molecular components in response to external or genetic perturbations.

PMID:
28648677
DOI:
10.1016/j.ymeth.2017.06.023
[Indexed for MEDLINE]

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