DMS-Seq Library Preparation Protocol

Sequencing libraries were prepared as described (Rouskin et al., 2014 (link)). Specifically, DMS treated mRNA samples were denatured for 2 min at 95°C and fragmented at 95°C for 2 min in 1x RNA fragmentation buffer (Zn²⁺ based, Ambion). The reaction was stopped by adding 1/10 vol of 10X Stop solution (Ambion) and quickly placed on ice. The fragmented RNA was run on a 10% TBU (Tris borate urea) gel for 60 min. Fragments of 60–70 nucleotides in size were visualized by blue light (Invitrogen, Carlsbad CA) and excised. Reverse transcription was performed in a 20 µL volume at 52°C using Superscript III (Invitrogen), and truncated reverse transcription products of 25–45 nucleotides were extracted by gel purification.