Northern Blot Analysis of Small and Large RNAs

Northern blots were performed using total RNA exactly as described previously (Melamed et al., 2020 (link)). For sRNAs, 5 μg of RNA were fractionated on 8% polyacrylamide urea gels containing 6 M urea (1:4 mix of Ureagel Complete to Ureagel-8 (National Diagnostics) with 0.08% ammonium persulfate) and transferred to a Zeta-Probe GT membrane (Bio-Rad). For longer RNAs, 10 μg of RNA were fractionated on a 2% NuSieve 3:1 agarose (Lonza), 1X MOPS, 2% formaldehyde gel and transferred to a Zeta-Probe GT membrane (Bio-Rad) via capillary action overnight. 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. Membranes were blocked in ULTRAhyb-Oligo Hybridization Buffer (Ambion) and hybridized with 5′ ³²P-end labeled oligonucleotides probes (listed in Supplementary Table 8). After an overnight incubation, the membranes were rinsed with 2X SSC/0.1% SDS and 0.2X SSC/0.1% SDS prior to exposure on film. Blots were stripped by two 7-min incubations in boiling 0.2% SDS followed by two 7-min incubations in boiling water.