Rnaseout

1 μg of NAD-RNA (38 nt) or m⁷Gppp-RNA (38 nt) was ligated with 5 μM 3′ adaptor oligo listed in Supplementary Table S5, in the presence of 10 U T4 RNA ligase 1 (New England Biolabs, catalog: M0202), 10% of PEG8000, 1 mM ATP and 40U RNaseOUT in 20 μl of 1x T4 RNA ligase buffer. Reaction was incubated at 16°C for 16 h. RNAs were purified by Trizol LS (Ambion, catalog: 10296010) according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold (Invitrogen, catalog: S11494) and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).

Run-off transcription was carried out to synthesize full-length ZIKV RNAs in vitro from a cloned cDNA of pZIKV or its derivatives [12 (link),61 (link)]. In short, each full-length ZIKV cDNA clone was first linearized by digestion with PsrI (SibEnzyme, West Roxbury, MA, USA). In all cases, ~200 ng of the linearized cDNA template was mixed in a 25-μL transcription reaction with 20 U SP6 RNA polymerase, 0.6 mM cap analog m⁷GpppA, 1 mM each NTP, 10 mM dithiothreitol, 40 U RNaseOUT, and the buffer supplied by the manufacturer (New England Biolabs, Ipswich, MA, USA). The reactions were first allowed to proceed at 37 °C for 1 h and further incubated for an additional 30 min with 10 U DNase I to degrade the template cDNA. RNA transcripts were quantified by adding 0.5 μM [³H]UTP (PerkinElmer, Boston, MA, USA) per reaction and calculating the amount of its incorporation into RNA based on the binding of RNA to DE81 paper (Whatman, Maidstone, United Kingdom). The integrity of the RNA transcripts was examined by running an aliquot of each reaction on a 0.6% agarose gel and visualizing the bands by ethidium bromide staining.