Quantitative profiling of SMG6 cleavage sites in HEK293 cells

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.