AI Article Synopsis
- Cell type-specific molecular profiling helps researchers understand the variety of cell types in complex tissues.
- Translating ribosome affinity purification (TRAP) is a technique that purifies actively translating mRNAs by using epitope-tagged ribosomal subunits, avoiding the limitations of traditional cell sorting.
- The protocol outlines steps to create transgenic animals with tagged ribosomes, prepare them for experiments, purify ribosome-mRNA complexes, and sequence the purified mRNA for further analysis.
Article Abstract
Cell type-specific molecular profiling is widely used to gain new insights into the diverse cell types that make up complex biological tissues. Translating ribosome affinity purification (TRAP) is a method in which the cell type-specific expression of epitope-tagged ribosomal subunits allows one to purify actively translating mRNAs without the need for cell sorting or fixation. We adapted this method for use in to identify novel transcripts in single cell types or to identify the effects of environmental changes on the transcriptomes of larger cohorts of cells. In this protocol, we describe methods to generate transgenic animals bearing tagged ribosomes in cells of interest, prepare these animals for immunoprecipitation, purify ribosome-mRNA complexes, and obtain purified mRNA for next-generation sequencing.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854165 | PMC |
| http://dx.doi.org/10.21769/BioProtoc.3328 | DOI Listing |
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