Northern Blot Analysis of Viral RNAs

Total RNA was extracted using the TRIreagent (Sigma), as previously described [37 (link)]. Ten micrograms of total RNA were separated on a denaturing 12% polyacrylamide gel (8M urea, 1 × MOPS buffer) and transferred to a Hybond NX membrane (Amersham Biosciences, Piscataway, NJ, USA) with semidry electroblotting [38 (link)]. RNA was crosslinked (1200 × 100 μJ/cm², 254 nm) to the membrane using a CL-1000 UV Crosslinker (UVP, CA, USA). The membrane was hybridized in an ULTRAhyb-Oligo buffer (Thermo Fisher, Waltham, MA, USA) with γ-³²P-ATP labelled hybridization probes detecting 5′ and 3′ ends of the HAdV-4 VA RNAI, HAdV-5 VA RNAI, or tRNA-Lysine (Supplementary Table S2). After overnight hybridization, the membrane was washed 3× for 10 min at 42 °C in a 3× SSC, 0.5% SDS buffer followed by a single wash with a 1× SSC, 0.5% SDS buffer for 15 min at 42 °C. Radioactive signals were detected using a Pharos FX^TM Plus Molecular Imager and analysed using a Quantity One 4.6.9 (Bio-Rad).