Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes—GMUCT 2.0

The advent of high-throughput sequencing has led to an explosion of studies into the diversity, expression, processing, and lifespan of RNAs. Recently, three different high-throughput sequencing-based methods have been developed to specifically study RNAs that are in the process of being degraded. All three methods—genome-wide mapping of uncapped and cleaved transcripts (GMUCT), parallel analysis of RNA ends (PARE), and degradome sequencing—take advantage of the fact that Illumina sequencing libraries use T4 RNA ligase 1 to ligate an adapter to the 5′ end of RNAs that have a free 5′-monophosphate. This condition for T4 RNA ligase 1 substrates means that mature mRNAs are not substrates of the enzyme because they have a 5′-cap moiety. As a result, these sequencing libraries are specifically made up of clones of decapped or degrading mRNAs resulting from 5′-to-3′ or nonsense-mediated decay (NMD) and the 3′ fragment of cleaved microRNA (miRNA) and small interfering RNA (siRNA) target RNAs. Here, we present a massively streamlined protocol for GMUCT that takes 2–3 days, can be initiated with as little as 5 μg of starting total RNA, and involves only one gel size-selection step. We show that the resulting datasets are similar to those produced using the previous GMUCT and PARE protocols. In total, our results suggest that this method will be the preferable approach for future studies of RNA degradation intermediates and small RNA-mediated cleavage in eukaryotic transcriptomes.