This protocol provides a detailed explanation of of the steps necessary for successful Direct RNA Library preparation for Oxford Nanopore Sequencing. The protocol explains the steps needed for RNA sample preparation based on TRIzol extraction and Poly(A)Purist Mag kit enrichment prior to Direct RNA library preparation protocol. The library preparation protocol is based on the Library preparation protocols for RNA-002 kits, yet offers additional advice on what we think is important for a successful library with minimal RNA degradation.

The protocol is used to assess poly(A) tail length using the tailfindr package. The poly(A) tail is a homopolymeric stretch of adenosine at the 3`-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. With the introduction of native RNA sequencing by Oxford Nanopore Technologies (ONT), it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making genome-wide transcript-specific poly(A) tail length assessment in native RNA feasible. For more information on tailfindr visit the publication or the GitHub repository