Poly(A) Profiling for Euglenozoans Using Oxford Nanopore Direct RNA Sequencing, Illustrated by the Example of Euglena...

There are numerous methods to study polyadenylation. These techniques offer insights into mRNA abundance and the dynamic regulation of poly(A) tail length and nucleotide content. The Oxford Nanopore Technologies (ONT) platform is particularly noteworthy among them. It allows analysis of the whole transcriptome at the single-molecule level without the amplification biases introduced by PCR-based methods.Most current studies in this area have focused on well-established model organisms such as humans, yeast, Arabidopsis, and Caenorhabditis elegans. In contrast, the poly(A) dynamics remains largely unexplored in non-model organisms like euglenids. These organisms possess a range of unique evolutionary traits, and investigating their poly(A) metabolism may elucidate the evolution of the enzymatic machinery that controls mRNA processing, stability, and turnover.Here, we present a step-by-step protocol for measuring poly(A) tail lengths and characterizing the nucleotide makeup of poly(A) tails in Euglena gracilis using ONT Direct RNA Sequencing.