GSM4787260: DMS-MaPseq analysis of SARS-CoV-2, replicate #1; Severe acute respiratory syndrome coronavirus 2; OTHER
Approximately 5x106 of the harvested infected cells were resuspended in 1 mL of TriPure Isolation Reagent (Sigma Aldrich, cat. 11667157001) and 200 μl of chloroform were added. The sample was vigorously vortexed for 15 sec and then incubated for 2 min at room temperature, after which it was centrifuged for 15 min at 12,500 x g (4°C). The upper aqueous phase was collected in a clean 2 mL tube, supplemented with 1 mL (~2 volumes) of 100% ethanol, and then loaded on an RNA Clean & Concentrator-25 column (Zymo Research, cat. R1017). In vitro folding was carried out as previously described5,10. Briefly, ~5 μg of total RNA from infected Vero E6 cells was first depleted of ribosomal RNAs using the RiboMinus™ Eukaryote System v2 (ThermoFisher Scientific, cat. A15026), following manufacturer instructions. Ribo- RNA in a volume of 39 μl was denatured at 95°C for 2 min, then transferred immediately to ice and incubated for 1 min. 10 μl of ice-cold 5X RNA Folding Buffer [500 mM HEPES pH 7.9; 500 mM NaCl] supplemented with 20 U of SUPERase•In™ RNase Inhibitor (ThermoFisher Scientific, cat. AM2696) were added. RNA was then incubated for 10 min at 37°C to allow secondary structure formation. Subsequently, 1 μl of 500 mM MgCl2 (pre-warmed at 37°C) was added and RNA was further incubated for 20 min at 37°C to allow tertiary structure formation. For probing of RNA, DMS was pre-diluted 1:6 in 100% ethanol and added to a final concentration of 150 mM. Samples were then incubated at 37°C for 2 min. Reactions were then quenched by the addition of 1 volume DTT 1.4 M and then purified on an RNA Clean & Concentrator-5 column (Zymo Research, cat. R1013). DMS-MaPseq of SARS-CoV-2 was conducted a previously described, with minor changes. First, probed RNA was fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 8 μl NF H2O. Eluted RNA was supplemented with 1 μl 50 μM random hexamers and 2 μl dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 20 μl. Reactions were supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 1 μl DTT 0.1 M, 20 U SUPERase•In™ RNase Inhibitor and 200 U TGIRT™-III Enzyme (InGex, cat. TGIRT50). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 57°C. TGIRT-III was degraded by addition of 2 μg Proteinase K, followed by incubation at 37°C for 20 min. Proteinase K was inactivated by addition of Protease Inhibitor Cocktail (Sigma Aldrich, cat. P8340). Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, following manufacturer instructions.