GSM5696673: SHAPE-MaP analysis of SARS-CoV-2 (2A3 treatment); Severe acute respiratory syndrome coronavirus 2; OTHER
To 1 mL of infected cells in QIAzol Lysis Reagent, 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 a Monarch® RNA Cleanup Kit column (NEB, cat. T2030L). In vitro folding was carried out as previously described10,22. Briefly, ~5 μg of total RNA from infected cells was first depleted of ribosomal RNAs using the RiboMinus™ Eukaryote System v2 (ThermoFisher Scientific, cat. A15026), as per manufacturer instructions. rRNA-depled RNA in a volume of 39 μl was denatured at 95°C for 2 min, then transferred to ice 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 15 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 15 min at 37°C to allow tertiary structure formation. For probing of RNA, 2A3 was added to a final concentration of 100 mM (assuming a stock concentration of 1 M). An equal volume of DMSO was added to the control samples. Samples were then incubated at 37°C for 5 min. Reactions were quenched by the addition of 1 volume DTT 1 M and then purified on Monarch® RNA Cleanup Kit columns. SHAPE-MaP library preparation was conducted as previously described, with minor changes. Probed RNA was first 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 on Monarch® RNA Cleanup Kit columns and eluted in 8 μl NF H2O. Eluted RNA was end repaired by treatment with 1 U of rSAP (NEB, cat. M0371L) at 37°C for 30 min, plus 5 min at 70°C to heat-inactivate the enzyme, followed by treatment with 20 U of T4 PNK (NEB, cat. M0201L), in the presence of 1 mM ATP, at 37°C for 1 hour. 50 ng of end-repaired RNA was first ligated to 10 pmol of a pre-adenylated 3' adapter (rApp-AGATCGGAAGAGCACACGTCT-SpC3) using 200 U of T4 RNA Ligase 2 truncated KQ (NEB, cat. M0373L), in the presence of 12.5% PEG-8000, for 1 hour at 25°C. 10 pmol of a 5' RNA adapter (CUACACGACGCUCUUCCGAUCU) was then ligated using 30 U of T4 RNA Ligase 1 (NEB, cat. M0437M), in the presence of 1 mM ATP and 8% PEG-8000, at 25°C for 1 hour. The adapter-ligated RNA was then supplemented with 10 pmol of RT primer (AGACGTGTGCTCTTCCGATCT), 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 10 μl. Reactions were supplemented with 2 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 1 μl DTT 0.1 M, 0.5 μl dNTPs 10 mM, 20 U SUPERase•In™ RNase Inhibitor and 100 U SuperScript II (Thermo Fisher Scientific, cat. 18064014). Reactions were incubated at 42°C for 2 hours. RNA was degraded by addition of 1 μl NaOH 5 N, followed by incubation at 95°C for 3 min. After purification, barcodes were introduced by PCR, using NEBNext® High-Fidelity 2X PCR Master Mix (NEB, cat. M0541L) and NEBNext® Multiplex Oligos for Illumina® Dual-Index primers (NEB, cat. E7780S).