Quantification of Poly(A) Tail Length and Terminal Modifications Using Direct RNA Sequencing.

Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to measure poly(A) tail length allow for the study of selected transcripts, the advent of long-read sequencing technologies enabled the development of simple and robust protocols to measure poly(A) tail length at the transcriptome level. Here, we describe a direct RNA sequencing protocol to capture poly(A) tail terminal additions based on the splint ligation of barcoded oligos compatible with terminal guanylation and uridylation. We cover how to prepare the libraries and perform the bioinformatics analysis to simultaneously determine the length of the transcripts' poly(A) tails and detect the presence of terminal guanylation and uridylation.