SHAPE-MaP was performed essentially as previously described (Smola et al., Nat Protoc: 2015; 10.1038/nprot.2015.103). DMSO or 2A3-treated RNA was treated with 4 units of TURBO DNase (Invitrogen, AM2238) with the included buffer for 1 hour at 37 C. DNase-treated RNA was then purified using the Monarch Spin RNA Cleanup kit (New England Biolabs, T2030L). RNA was eluted in 13 l of nuclease-free water. RNA (10 l) was then combined with 1 l of 2 M reverse-transcription primer (5- AGA ACC TGT TGA TTC AAC AGC AC-3), heated to 65 C for 5 minutes, then immediately placed on ice. MaP buffer and SuperScript II Reverse Transcriptase (Invitrogen, 18064014) were then added, mixed, and incubated at 37 C for 3 hours. The reverse transcriptase was then heat inactivated at 70 C for 5 minutes and the cDNA products were purified using Illustra MicroSpin G-25 Columns. Library preparation was performed by first PCR amplifying the sfRNA using Phusion High-fidelity DNA Polymerase (New England Biolabs, M0530L) and flavivirus-specific primers (Forward: 5- AGA AGT CAG GCT GGA TTA AG-3; Reverse: 5- AGA ACC TGC TGA TTC AAC-3) and 5 l of cDNA template in a 50 l volume. PCR amplicons (5 l) were electrophoresed on a 1% agarose TAE gel and products were visualized using RedSafe nucleic acid staining solution. Libraries were constructed with the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096) and Illumina DNA/RNA UD Indexes with 1 ng cDNA (tagmented) as input. The final amplified libraries were purified using AMPure XP bead cleanup (Beckman Coulter, A63882). Libraries were validated by capillary electrophoresis on a TapeStation 4200 (Agilent), pooled at equimolar concentrations, and sequenced with PE100 reads on an NovaSeq 6000 (Illumina), targeting a depth of 500,000 reads per sample.