DMS-MaP on SARS-CoV2 FSE
In vitro RNA chemical Probing: Various SARS-CoV-2 FSE constructs were synthesized as G-blocks from Integrated DNA Technologies (IDT). This whole construct was flanked at both 5 and 3 end by RNA cassettes. T7 promoter region was added to the 5 end of each construct for in vitro transcription of RNA using T7 RNA polymerase using T7 HiScribe RNA synthesis kit (New England Biolabs). Synthesized RNA was subjected to DNase treatment (TURBODNase) and was further purified using Purelink RNA mini kit (Invitrogen) and quantified using nanodrop. For chemical probing, 6 g of purified in vitro transcribed RNA was denatured at 65 C for 5 min and snap-cooled in ice. Following denaturation, folding buffer (100 mM KCl, 10 mM MgCl2, 100 mM Bicine, pH 8.3) was added to the denatured RNA and the whole reaction was incubated at 37 C for 10 min. The folded RNA was further treated with 10 ul of 1:10 ethanol diluted Dimethyl Sulfate (DMS). For control, equivalent volume of ethanol was added to the folded RNA. Probing was initiated by incubating the reaction mixture at 37 C for 5 min and was quenched afterwards by adding 100 ul of 20% -mercaptoethanol (-ME). Modified and un-modified RNAs were purified using Purelink RNA mini kit and quantified using nanodrop. Library construction, sequencing, and data processing:Following chemical probing, both modified and un-modified RNA were reverse transcribed using specific primers  complementary to 3 RNA cassette and Superscript II reverse transcriptase under error prone conditions. The generated cDNA was purified using G50 column (GE healthcare) and subjected to second strand synthesis (NEBNext Second Strand Synthesis Module). For constructing next-generation sequencing libraries, the double-stranded (ds) cDNA was PCR amplified with primers directed against 5 and 3 RNA cassettes and NEB Q5 HotStart polymerase (NEB). To introduce unique barcodes secondary PCR was performed using TrueSeq primers (NEB). Amplified products were purified using Ampure XP (Beckman Coulter) beads and quantification of the libraries were done using Qubit dsDNA HS Assay kit (ThermoFisher). Purified libraries were quality checked using Agilent Bioanalyzer. These libraries were finally sequenced as 2 x 151 paired end read on Illumina MiSeq platform.