Pairs of RNA molecules transcribed from partially or entirely complementary loci are called cis-natural antisense transcripts (cis-NATs), and they play key roles in the regulation of gene expression in many organisms. A promising experimental tool for profiling sense and antisense transcription is strand-specific RNA sequencing (ssRNA-seq). To identify cis-NATs using ssRNA-seq, we developed a new computational method based on a model comparison framework that incorporates the inherent variable efficiency of generating perfectly strand-specific libraries. Applying the method to new ssRNA-seq data from whole-root and cell-type"specific Arabidopsis libraries confirmed most of the known cis-NAT pairs and identified 918 additional cis-NAT pairs. Newly identified cis-NAT pairs are supported by polyadenylation data, alternative splicing patterns, and RT-PCR validation. We found 209 cis-NAT pairs that have opposite expression levels in neighboring cell types, implying cell-type"specific roles for cis-NATs. By integrating a genome-wide epigenetic profile of Arabidopsis, we identified a unique chromatin signature of cis-NATs, suggesting a connection between cis-NAT transcription and chromatin modification in plants. An analysis of small-RNA sequencing data showed that 1/44% of cis-NAT pairs produce putative cis-NAT"induced siRNAs. Taken together, our data and analyses illustrate the potential for multifaceted regulatory roles of plant cis-NATs. [ABSTRACT FROM AUTHOR]