Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically-derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analyses. We produced DRS datasets using modification-free transcripts from in vitro transcription of cDNA from six immortalized human cell lines. We characterized variation across cell lines and demonstrated how these may be interpreted. These data will serve as a versatile control and resource to the community for RNA modification analyses of human transcripts.
