RNA fragmentation

Crosslinked RNAs were fragmented using ShortCut RNase III (NEB, M0245). Briefly, 10 μg of crosslinked RNA was fragmented using 10 μl of RNase III with 50 mM MnCl₂ and 1× supplied shortcut buffer at 37 °C for 5 min. After fragmentation, equal volume of phenol was immediately added to stop the reaction. Then one-tenth volume of 3 M sodium acetate (pH 5.3), 3 μl of GlycoBlue (Invitrogen, AM9516), three volume of pure ethanol were added to precipitate RNA. Fragmented RNA was resuspended in RNase-free water and checked size distribution using TapeStation. Different fragmentation conditions also were tested in this study, such as different RNase III amount, different fragmentation time, and different concentration of MnCl₂.

After 5 min of shortcut digestion, reaction need to be stopped as soon as possible to get the optimal size distribution. Longer reaction time will reduce the RNA fragments size. The size distribution of fragmented crosslinked RNA can be analyzed by Bioanalyzer or TapeStation system (Agilent TapeStation Software v3.2). If using TapeStation, high-sensitivity D1000 ScreenTape plus high-sensitivity RNA sample buffer should be used because of short RNA size after fragmentation.