Direct RNA sequencing enables single-nucleotide m6A detection in endogenous transcript isoforms

We report the direct RNA sequencing of HEK293 and a primary human mammary epithelial cell (HMEC) line using Oxford Nanopore based sequencing. Using this data, we built an algorithm to detect m6A modifications within the DRACH motif context. Evaluation of m6A sites was carried out with HEK METTL3 knockdown and HMEC ALKBH5 over expression cell lines. Total RNA was isolated from HEK and HMEC cell lines using Trizol or Direct-zol, respectively. RNA was then poly-A selected using oligo-dT magnetic resin before library preparation using Oxford Nanopore’s direct RNA-sequencing kit and protocol. Sequencing output was mapped to reference genomes and resquiggled using Tombo. Tombo’s fraction modified and coverage files were subsequently combined and used as input for m6A detection.