Comprehensive profiling of mRNA polyadenylation in specific cell types in vivo by cTag-PAPERCLIP

Abstract

The ability to generate cell-type specific mRNA polyadenylation (pA) maps from complex tissues is crucial for understanding how alternative polyadenylation (APA) is regulated in individual cell types in their physiological environment under different conditions. In this chapter, we discuss cTag-PAPERCLIP, a recently developed method combining the well-established CLIP (crosslinking immunoprecipitation) technique and the Cre-lox system to achieve customized cell-type specific APA profiling from mouse tissue without cell purification or enrichment. In cTag-PAPERCLIP, selective expression of GFP-tagged poly(A) binding protein (PABP-GFP) in the desired cell type is achieved through Cre-mediated activation of a latent knock-in allele of PABP-GFP. Immunoprecipitation of PABP-GFP then allows mRNA 3' end fragments in the desired cell type to be specifically retrieved from ultraviolet (UV)-irradiated whole tissue lysate. The mRNA fragments are subsequently turned into a cDNA library to provide a comprehensive APA map and an mRNA expression profile of the chosen cell type through deep sequencing.

Keywords: CLIP-seq; Cell-type specificity; Crosslinking immunoprecipitation; RNA processing; mRNA polyadenylation; poly(A)-binding protein.