Jade Forster from the University of Southampton in the UK presented at London Calling 2023 about “Identifying m6A RNA modifications in neuroblastoma cell lines using nanopore sequencing.” This session was timely as we are doing our first direct RNA sequencing experiment and GridION run! Forster spoke about epitranscriptomics and RNA modifications. They noted that the most common modification is N6-methyladenosine (m6A) and has a role in mRNA splicing, translation, and stability of transcripts. There are readers, writeres, and erasers of m6A modifications. Forster told the audience that neuroblastoma and the epitranscriptome have important connections! With traditional technologies, RNA had to be converted to cDNA before sequencing. Oxford Nanopore Technologies offer the opportunity to interpret different signals from modified RNA bases. Forster explained that from one experiment there can be transcriptomics information, full length isoforms, splicing, differential gene expression, polyA tail analysis… and more! Forster and team used the GridION device and R9.4.1 flow cells with the direct RNA sequencing kit. They used MinKNOW, minimap2, Nanopolish, event align, and m6anet. They grew cell cultures from high-risk neuroblastoma cell lines and extracted RNA using Trizol. They used total RNA because enrichment for polyA RNA did not yield enough. They sequenced each cell line over four flow cells for each cell line. They obtained N50s of 1,571 and 1, 730 for the two cell lines and ~2 million reads for each. They then identified significantly enriched biological processes. Forster concluded that they used direct RNA sequencing on two human high-risk neuroblastoma cell lines to identify m6A modifications using m6anet. They identified enrichment in biological processes such as histone H3K36 methylation and will continue investigating/analyzing other modifications. This session was very relevant to what we are doing in the lab with bacterial transcriptomics! I’m excited about what we can identify… what is the significance of m6A modifications in bacteria?
