Fractionation and Detection of RNAs

For smaller RNAs, total RNA (5 μg) was separated on a denaturing 8% polyacrylamide urea gel containing 6 M urea (1:4 mix of Ureagel Complete to Ureagel-8 (National Diagnostics) with 0.08% ammonium persulfate) in 1 X TBE buffer at 300 V for 90 min. The RNA was transferred to a Zeta-Probe GT membrane (Bio-Rad) at 20 V for 16 hr in 0.5 X TBE. For longer RNAs, total RNA (10 μg) was fractionated on formaldehyde-MOPS agarose gels as previously described (Adams et al., 2017 (link)). Briefly, RNA was denatured in 3.7% formaldehyde (Fisher), 1 X MOPS (20 mM MOPS, 5 mM NaOAc, 1 mM EDTA, pH 7.0) and 1 X RNA loading dye (Thermo Fisher Scientific) for 10 min at 70 °C and incubated on ice. The RNA was loaded onto a 2% NuSieve 3:1 agarose (Lonza), 1 X MOPS, 2% formaldehyde gel and separated at 125–150 V at 4 °C for 1–2 hr and then transferred to a Zeta-Probe GT membrane (Bio-Rad) via capillary action overnight (Streit et al., 2009 (link)). For both types of blots, the RNA was crosslinked to the membranes by UV irradiation. RiboRuler High Range and Low Range RNA ladders (Thermo Fisher Scientific) were marked by UV-shadowing. Oligonucleotide probes (listed in Supplementary file 3) for the different RNAs were labelled with 0.3 mCi of [γ-³²P] ATP (Perkin Elmer) by incubating with 10 U of T4 polynucleotide kinase (New England Biolabs) at 37 °C for 1 hr.