The article presents a novel method for strand-specific sequencing of complementary DNA (ssRNA-Seq) to preserve information about the direction of transcripts. This approach involves incorporating deoxy-UTP during second-strand cDNA synthesis, followed by selective destruction of the uridine-containing strand using uracil-N-glycosylase (UNG). The method is demonstrated using Saccharomyces cerevisiae and mouse brain transcriptomes, showing that it accurately determines transcript orientation, gene structure, and expression levels. The orientation of transcripts is crucial for understanding gene function, identifying new genes, and studying promoter-associated and antisense transcription. The authors also discuss the reproducibility of the method and its advantages over other transcriptome analysis techniques, such as microarrays and SAGE. The ssRNA-Seq data and a genome browser for visualization are available online.The article presents a novel method for strand-specific sequencing of complementary DNA (ssRNA-Seq) to preserve information about the direction of transcripts. This approach involves incorporating deoxy-UTP during second-strand cDNA synthesis, followed by selective destruction of the uridine-containing strand using uracil-N-glycosylase (UNG). The method is demonstrated using Saccharomyces cerevisiae and mouse brain transcriptomes, showing that it accurately determines transcript orientation, gene structure, and expression levels. The orientation of transcripts is crucial for understanding gene function, identifying new genes, and studying promoter-associated and antisense transcription. The authors also discuss the reproducibility of the method and its advantages over other transcriptome analysis techniques, such as microarrays and SAGE. The ssRNA-Seq data and a genome browser for visualization are available online.