Northern Blot Analysis of Viral Small RNAs

Northern blot analysis was conducted as described previously [29 (link),40 (link)]. In short, total RNA was isolated from the SFV or CHIKV infected Aag2 cells (2×10⁶ cells, MOI 10, 48 hpi) by using Trizol reagent. 15 µg of RNA was size-fractioned on a 0.5× TBE, 7 M Urea, 15% polyacrylamide gel, thereafter transferred to Hybond NX nylon membranes (GE Healthcare, Chicago, IL, USA). RNA was chemically cross-linked to membrane by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma, St. Louis, MO, USA). SFV or CHIKV-specific small RNAs were probed with a set of DNA oligonucleotides (Supplementary Table S1) that were 5′ end-labelled with [³²P] γ-adenosine-triphosphate (Perking Elmer, Waltham, MA, USA) using T4 Polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Hybridization of membranes with oligonucleotide-probes was performed overnight at 42 °C in Ultrahyb Oligo hybridization buffer (Thermo Fisher Scientific). Membranes were washed twice at 42 °C with each of the following three buffers: 2× SSC and 0.5% SDS, 2× SSC and 0.2% SDS, 0.2× SSC and 0.2% SDS. The membrane was exposed to a phosphoimager screen for signal detection and quantification.