3' End sequencing of pA⁺ and pA^- RNAs

The identity and metabolism of RNAs are often governed by their 5' and 3' ends. Single gene loci produce a variety of transcript isoforms, varying primarily in their RNA 3' end status and consequently facing radically different cellular fates. Knowledge about RNA termini is therefore key to understanding the diverse RNA output from individual transcription units. In addition, the 3' end of a nascent RNA at the catalytic center of RNA polymerase provides a precise and strand-specific measure of the transcription process. Here, we describe a modified RNA 3' end sequencing method, that utilizes the in vivo metabolic labeling of RNA followed by its purification and optional in vitro polyadenylation to provide a comprehensive view of all RNA 3' ends. The strategy offers the advantages of (i) nucleotide resolution mapping of RNA 3' ends, (ii) increased sequencing depth of lowly abundant RNA and (iii) inference of RNA 3' end polyadenylation status. We have used the method to study RNA decay and transcription termination mechanisms with the potential utility to a wider range of biological questions.