Our method, RNAtag-Seq, utilizes a molecular barcoding strategy that generates multiple libraries in a single reaction, lowers labor and reagent costs per sample, and produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches. Barcoding in RNAtag-Seq is achieved through direct ligation of adaptors to RNA, enabling high quality, strand-specific, quantitative sequencing of full-length transcripts in diverse prokaryotic and eukaryotic species.