Short read direct RNA-sequencing of mammalian bone marrow derived macrophages for chimeric RNA discovery

Eukaryotic mRNA is classically expected to be monocistronic, linear, and encoded by a single defined genetic locus. Conversely, emerging evidence has identified bicistronic translation and circularized mRNA as pervasive unconventional features of mammalian transcriptomes. During oncogenic translocation in humans, DNA from distinct genes can fuse together and encode chimeric mRNAs that directly contribute to tumorigenesis. However, it remains widely unknown whether or not chimeric mRNAs exist in the absence of chromosomal rearrangements, and if they contribute to normal physiological processes. To assay for chimeric mRNA (chRNA) expression in mammalian innate immune cells while circumventing chRNA detection limitations associated with conventional RNA-sequencing (RNA-seq) technology, including read fragmentation and artifact generation by template switching, we performed long read direct RNA sequencing (LR-DRS) of differentially polarized bone marrow-derived macrophages (BMDMs) using the Oxford Nanopore platform, and complemented this with conventional short-read RNA-sequencing.