The ability to map transcription start sites is critical for studies of gene regulation and for identification of novel RNAs. Conventional RNA-seq is often insufficient for identification of transcription start sites due to low coverage and/or RNA processing events. We have developed a highly sensitive, genome-scale method for detection of transcription start sites in bacteria. This method uses deep sequencing of cDNA libraries to identify transcription start sites with strand specificity at nucleotide resolution. Here, we describe the application of this method for transcription start site identification in Escherichia coli.