Cell Type-specific mRNA Purification in Caenorhabditis elegans via Translating Ribosome Affinity Purification

Ian G McLachlan; Steven W Flavell

doi:10.21769/BioProtoc.3328

Cell Type-specific mRNA Purification in Caenorhabditis elegans via Translating Ribosome Affinity Purification

Bio Protoc. 2019 Aug 5;9(15):e3328. doi: 10.21769/BioProtoc.3328.

Authors

Ian G McLachlan¹, Steven W Flavell¹

Affiliation

¹ Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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 C. elegans 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.

Keywords: Caenorhabditis elegans; Expression profiling; RNA purification; RNA-seq; Ribosome profiling.