Quantitative profiling of SMG6 cleavage sites in HEK293 cells

The nonsense-mediated mRNA decay pathway targets specific mRNAs for degradation in a translation-dependent manner. NMD can induce RNA turnover via either the bulk mRNA deadenylation and decapping machinery or via the specialized SMG6 endonuclease. SMG6 is a magnesium-dependent nuclease, leading to the presence of 5' phosphates on cleavage intermediates. These 5' phosphates can be captured by adapter ligation for identification by high-throughput sequencing. Here we adapt Akron5-seq for quantitative identification of SMG6 cleavage sites in HEK293 cells. This approach improves upon previous methods for SMG6 cleavage site mapping by using 1) RNA fragmentation and 3' adapter ligation to avoid length biases caused by low reverse transcriptase processivity, 2) unique molecular identifiers to remove PCR duplicates, and 3) triplicate measurements to enable robust statistical analyses.