Nanopore long-read RNA-seq and absolute quantification delineate transcription dynamics in early embryo development of an insect pest

Abstract The Oxford Nanopore Technologies’ long-read RNA sequencing (RNAseq) platform can yield full length transcripts that can be very instrumental in improving the characterization of non-model organisms. The resolution of RNAseq can be increased by addition of external RNA molecules of known concentration such as ERCC in order to obtain absolute gene expression quantification. This protocol details the procedure to use ONT long-read RNAseq with addition of ERCC external RNAs. This protocol can be used with total RNA extracted from any source for which transcripts with a poly(A) tail at the 3’ end are the target. The protocol should be possible to complete in a single day (12 hours) followed by sequencing which takes another 48 hours.