This study presents a strand-specific RNA-Seq method that preserves information about the direction of transcripts. The method involves incorporating deoxy-UTP during the second strand cDNA synthesis, followed by the destruction of the uridine-containing strand in the sequencing library, allowing the identification of transcript orientation. This approach enables more accurate determination of gene structure and expression, identification of new genes, and study of promoter-associated and antisense transcription. The method was tested using Saccharomyces cerevisiae and mouse brain transcriptomes, and the results are available online. The RNA-Seq procedure is simple, has a large dynamic range and high sensitivity, and can unequivocally identify splicing and RNA editing products as well as allele-specific transcripts. RNA-Seq provides several advantages over previous high-throughput approaches, including microarray hybridization, gene-specific and tiling arrays or SAGE analyses. Unlike SAGE, RNA-Seq does not depend on the presence of particular restriction sites within the cDNA. The depth of RNA-Seq analysis is flexible, providing a dynamic range typically an order of magnitude greater than one can achieve with hybridization arrays. The digital character of the RNA-Seq data permits to compare and pool results from different laboratories. No prior information about transcript sequences is required, allowing detection of novel transcripts. It is possible to estimate the absolute level of gene expression and to study the structure of transcripts. A weakness of RNA-Seq is the inability to determine the polarity of RNA transcripts without laborious modification of the protocol. The polarity of the transcript is important for correct annotation of novel genes, because it provides essential information about the possible function of a gene, both at the RNA (structure and hybridization to other nucleic acid molecules) and protein levels. In addition, many genomic regions give rise to transcripts from both strands. Antisense transcription is characteristic for eukaryotic genes and is thought to play an important regulatory role. Overlapping genes are common for compact genomes of prokaryotes and lower eukaryotes. Knowledge of a transcript's orientation helps to resolve colliding transcripts and to correctly determine gene expression levels in the presence of antisense transcripts. The study describes a simple modification of RNA-Seq method that addresses this problem. Incorporation of deoxy-UTP during the second strand cDNA synthesis and subsequent destruction of the uridine-containing strand in the sequencing library allowed us to identify the orientation of transcripts. The method was illustrated using sequencing data for yeast and mouse transcriptomes. The results show that the ssRNA-Seq method is highly reproducible and provides accurate information about gene expression and structure. The method is compatible with commercially available kits and can be adapted for other second generation sequencing platforms. The study also highlights the importance of transcript orientation in determining gene expression levels, identifying new genes, and studying promoter-associated and antisense transcription. The results are available online and the data are submitted to the NCBI Short Read Archive.This study presents a strand-specific RNA-Seq method that preserves information about the direction of transcripts. The method involves incorporating deoxy-UTP during the second strand cDNA synthesis, followed by the destruction of the uridine-containing strand in the sequencing library, allowing the identification of transcript orientation. This approach enables more accurate determination of gene structure and expression, identification of new genes, and study of promoter-associated and antisense transcription. The method was tested using Saccharomyces cerevisiae and mouse brain transcriptomes, and the results are available online. The RNA-Seq procedure is simple, has a large dynamic range and high sensitivity, and can unequivocally identify splicing and RNA editing products as well as allele-specific transcripts. RNA-Seq provides several advantages over previous high-throughput approaches, including microarray hybridization, gene-specific and tiling arrays or SAGE analyses. Unlike SAGE, RNA-Seq does not depend on the presence of particular restriction sites within the cDNA. The depth of RNA-Seq analysis is flexible, providing a dynamic range typically an order of magnitude greater than one can achieve with hybridization arrays. The digital character of the RNA-Seq data permits to compare and pool results from different laboratories. No prior information about transcript sequences is required, allowing detection of novel transcripts. It is possible to estimate the absolute level of gene expression and to study the structure of transcripts. A weakness of RNA-Seq is the inability to determine the polarity of RNA transcripts without laborious modification of the protocol. The polarity of the transcript is important for correct annotation of novel genes, because it provides essential information about the possible function of a gene, both at the RNA (structure and hybridization to other nucleic acid molecules) and protein levels. In addition, many genomic regions give rise to transcripts from both strands. Antisense transcription is characteristic for eukaryotic genes and is thought to play an important regulatory role. Overlapping genes are common for compact genomes of prokaryotes and lower eukaryotes. Knowledge of a transcript's orientation helps to resolve colliding transcripts and to correctly determine gene expression levels in the presence of antisense transcripts. The study describes a simple modification of RNA-Seq method that addresses this problem. Incorporation of deoxy-UTP during the second strand cDNA synthesis and subsequent destruction of the uridine-containing strand in the sequencing library allowed us to identify the orientation of transcripts. The method was illustrated using sequencing data for yeast and mouse transcriptomes. The results show that the ssRNA-Seq method is highly reproducible and provides accurate information about gene expression and structure. The method is compatible with commercially available kits and can be adapted for other second generation sequencing platforms. The study also highlights the importance of transcript orientation in determining gene expression levels, identifying new genes, and studying promoter-associated and antisense transcription. The results are available online and the data are submitted to the NCBI Short Read Archive.