Northern Blot Analysis of Small RNAs

RNA was isolated as described previously [24 (link)]. RNA samples (10 μg) were denatured in RNA loading buffer (95% (v/v) formamide, 0.1% (w/v) xylene cyanol, 0.1% (w/v) bromophenol blue, 10 mM EDTA) for 5 min at 95°C. They were then separated on 6% acrylamide, 8.3 M urea gels, and transferred to Hybond XL membranes (GE Healthcare) by semi-dry blotter at 25 V for 1 h (Biorad). RNA was UV crosslinked and pre-conditioned in Rapid Hybridisation buffer (GE Healthcare) at 42°C for 1 h, before 5' [γ-32P] end-labeled probes for CjNC1 or CjNC4 were added and incubated for 16 h. Probe sequences are given in S1 Table. Membranes were washed in 5 × SSC/0.1% SDS, 1 × SSC/0.1% SDS and 0.5 × SSC/0.1% SDS. Signals were visualized on a Typhoon 9200 phosphorimager (GE Healthcare) after at least 16 h exposure to a phosphor screen.