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Background  Bacteria rely on efficient gene regulatory mechanisms to switch between genetic programs when they are facing new environments. Although this regulation can occur at many different levels, one of the key steps is the initiation of transcription. Identification of the first nucleotide transcribed by the RNA polymerase is therefore essential to understand the underlying regulatory processes, since this provides insight on promoter strength and binding sites for transcriptional regulators, and additionally reveals the exact 5’ untranslated region of the transcripts, which often contains elements that regulate translation.    Results  Here we present data from a novel TSS-EMOTE assay (Transcription Start Specific Exact Mapping Of Transcriptome Ends) to precisely map the transcription initiation sites of four entire transcriptomes. TSS-EMOTE is a variation of the dRNA-seq method, which has been combined with the EMOTE protocol, in order to increase detection of longer transcripts and limit biases introduced by PCR amplification of the Illumina sequencing library. Using TSS-EMOTE, 2018 promoters were detected in the opportunistic pathogen  Staphylococcus aureus , and detailed consensus sequences could be obtained for the RNA polymerase recognition elements (e.g. sigma factor binding sites). The data also revealed a 94 nt median length of the 5’ untranslated region in  S. aureus , giving important insights for future work on translational regulation. Additionally, the transcriptomes of three other opportunistic pathogens,  Staphylococcus epidermidis ,  Acinetobacter baumannii  and  Enterobacter aerogenes , were examined, and the identified promoter locations were then used to generate a map of the operon structure for each of the four organisms.    Conclusions  Mapping transcription start sites, and subsequent correlation with the genomic sequence, provides a multitude of important information about the regulation of gene expression, both at the transcriptional and translational level, by defining 5’ untranslated regions and sigma-factor binding sites. We have here mapped transcription start sites in four important pathogens using TSS-EMOTE, a method that works with both long and 3’-phosphorylated RNA molecules, and which incorporates Unique Molecular Identifiers (UMIs) to allow unbiased quantification.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-016-3211-3) contains supplementary material, which is available to authorized users.

TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens