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Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP)

Abstract

Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian CNS at a molecular level. To address this problem, we have recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell type–specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell type–specific mRNA profiles of any genetically defined cell type, and it has been used in organisms ranging from Drosophila melanogaster to mice and human cultured cells. Unlike other methodologies that rely on microdissection, cell panning or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and the potential artifacts that these treatments introduce) and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implement the TRAP methodology, which takes 2 d to complete once all materials are in hand.

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Figure 1: The translating ribosome affinity purification (TRAP) strategy.
Figure 2: Representative polyribosome profiles from fresh and frozen striatal tissue.

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Acknowledgements

We acknowledge members of the Greengard, Heintz and R. Darnell laboratories for their helpful discussions, advice and feedback. This work was supported by grants from The JPB Foundation, The Simons Foundation and National Institute of Mental Health (NIMH) award MH090963 to P.G., and by The Simons Foundation, The Howard Hughes Medical Institute and NIMH award MH090963 to N.H.

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M.H., R.K. and R.J.F. assembled the step-by-step protocol; M.H., N.H. and P.G. wrote the paper.

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Correspondence to Myriam Heiman.

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The authors declare no competing financial interests.

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Heiman, M., Kulicke, R., Fenster, R. et al. Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP). Nat Protoc 9, 1282–1291 (2014). https://doi.org/10.1038/nprot.2014.085

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