GSM4892123: DMSO, in vivo (SARS-CoV-2); Chlorocebus sabaeus; OTHER
200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min. Total RNA from infected and probed Vero E6 cells was first depleted of ribosomal RNAs using the RiboMinus™ Eukaryote System v2 (ThermoFisher Scientific, cat. A15026), following manufacturer instructions. RNA was then 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 1 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], 2 μl DTT 0.1 M, 1 μl MnCl2 120 mM, 20 U SUPERase•In™ RNase Inhibitor and 200 U SuperScript II RT. Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 42°C. SSII was heat-inactivated by incubating at 75°C for 15 min. 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. 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.