title: SHAPE-MaP of invitro SL1-4b-100nM OAS1: DMSO Rep1
design: RNAs used for SHAPE-MaP were cloned into a structural cassette and purified. Purified RNAs (2 ug) were heated to 75 C for 2 minutes and snap cooled on ice prior to refolding at 37 C for 30 minutes in 20 mM Tris-Cl (pH 7.4) and 7 mM MgCl2. RNAs were treated with 2A3 or DMSO at 37 C for 5 minutes and the reaction was quenched with 1 M DTT.  RNA was recovered using the NEB Monarch RNA clean-up kit and reverse transcribed with SuperScript II in a modified MaP buffer (25mM Tris-Cl (pH 8.0),187.5 mM KCl, 15 mM MnCl2, 25 mM DTT and 1.25 mM dNTPs) as previously described. After desalting (G-25 spin column, Cytiva), cDNA (5 l) was PCR amplified in a 50 l reaction using Phusion High-fidelity DNA Polymerase (New England Biolabs). For RNAs <200 nucleotides, amplicons were designed to include partial Nextera XT Illumina sequencing adapters and inline barcodes. Amplicons were purified using AMPure XP bead cleanup (Beckman Coulter) and quantified using a Qubit Flex (Invitrogen). Libraries were constructed with the Nextera XT DNA Library Preparation Kit (Illumina) and Illumina DNA/RNA UD Indexes, Tagmentation (Illumina) with 1 ng total cDNA as input (tagmentation was omitted for RNAs <200 nucleotides).