Knowledge of mitochondrial transcription start sites and promoter sequences is key to understanding mechanisms of transcription initiation in plant mitochondria. Transcription start sites can be straightforwardly determined by the mapping of primary transcript 5' ends. This chapter describes a next-generation sequencing-based protocol for the mitochondrial genome-wide mapping of transcription start sites in Arabidopsis thaliana. Like other strategies aiming at the determination of primary transcript 5' ends, this protocol exploits that only primary but not processed transcripts are 5'-triphosphorylated and, based on this property, can be enzymatically selected for. However, it uses nascent transcripts, in order to (1) enhance mitochondrial coverage compared with other compartments, (2) reduce rRNA and other background, and (3) also capture the primary 5' ends of rapidly degraded or processed transcripts.
Keywords: 5-Bromouridine labeling; 5′-GRO-seq; Arabidopsis; Mitochondria; Nascent RNA; Next-generation sequencing; RNA polymerase; Run-on transcription; Transcription start site.
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