Direct long-read RNA sequencing of Raji cells
To identify full-length cap-to-poly(A) mRNA isoforms of CD20 and rule out reverse transcription artifacts which are common in cDNA-seq approaches, long-read Oxford Nanopore direct RNA sequencing was performed on the Raji cell line. Overall design: From whole Raji cells, total RNA was isolated using the Maxwell® RSC simplyRNA Cells Kit (Promega). 500ng of mRNA was isolated from total RNA using Dyna-beads mRNA DIRECT kit (Invitrogen) and used for direct RNA (SQK-RNA002, ONT) library preparation. Subsequently, each library was loaded into a Spot-ON Flow Cell R9 version (FLO-MIN106D, ONT) and sequenced in a MinION Mk1B device (ONT) for 48 hours. Raw Fast5 files were converted to fastq with guppy (v3.4.5)