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The genomic and transcriptomic studies have been greatly accelerated by the next-generation sequencing technology. Currently, there are two main methods in transcriptomic studies, namely, Tiling microarray and RNA-seq. Comparatively, because of its overwhelming superiority in transcript coverage and resolution, as well as decrease of costs, RNA-seq has been employed in transcriptome analyses by an increasing number of research institutes. According to the mRNA enrichment or rRNA depletion methods, RNA-seq can be performed in six ways. Among them, dRNA-seq can descriminate the original transcripts from the processed RNAs based on a differential exonuclease treatment. This kind of method has been broadly applied in studies of prokaryotic transcriptional start sites, small regulatory RNAs, promoters and operons, etc. Here, we reviewed the detailed principle and technical process of dRNA-seq and its applications in prokaryotic transcriptome analyses. Besides, we also summarized the advantages and limitations of dRNA-seq at present stage, and then gave our perspective of its future development and potential applications, expecting to present useful references for civil researchers in related fields.