Mapping 5'-Ends and Their Phosphorylation State With EMOTE, TSS-EMOTE, and nEMOTE

Knowing the exact 5'-end of a specific RNA molecule has long been of great interest, since this information can reveal transcription start sites, which subsequently pinpoints promoter regions and potential binding sites for transcriptional regulators. Mapping of the 5'-end can also identify endoribonucleolytic cleavage sites, for the maturation of ribosomal RNA or events which initiate degradation of all or parts of the RNA molecule. If the 5' nucleotide of an RNA is triphosphorylated, then the nucleotide represents the original, unaltered 5'-end of the RNA. Similarly, if an endoribonuclease cleaves the RNA with a mechanism that leaves a monophosphate on the 5'-end downstream cleavage product, then it is useful to search exclusively for monophosphorylated 5'-ends to identify the cleavage sites of the endoribonuclease. This chapter details three variations of the EMOTE (exact mapping of transcriptome ends) protocol, which can identify RNA 5'-ends on a transcriptome-wide scale, and simultaneously provide information about the phosphorylation state of the detected ends. EMOTE, TSS-EMOTE (transcription start specific EMOTE), and nEMOTE (nonphosphorylated EMOTE) reveals mono-, tri-, and nonphosphorylated 5'-ends, respectively, and the protocols can be performed individually or in parallel on the same RNA sample.