Project: mRNA-seq on SARS-CoV-2-infected Calu-3 and Huh-7.5 cells; Cells: Huh-7.5; Treatment: Mock; Analysed data: See Manuscript Supp Tables 2 and 3
Cell culture and infection: 75,500 Huh-7.5 cells or 150,000 Calu-3 cells were seeded in each well of a 12-well plate with 1 mL media. Media: DMEM with 5% FBS and 1% NEAA for Huh-7.5 cells or EMEM (ATCC, 30-2003) with 10% FBS for Calu-3 cells. The next day, cells were infected by removing 500 L of media and adding 500 L of media with SARS-CoV-2 strain USA-WA1/2020 (BEI Resources, NR-52281) at 5,000 PFU/well (virus titer determined in Huh-7.5 cells). After one day, the wells were washed with PBS and cells were harvested in 1 mL TRIzol (Invitrogen, cat. 15596-018). N = 3 replicates (separate wells) per sample. RNA extraction: 2 ml MaXtract High Density tubes (Qiagen, 129056) were centrifugated at 12,00016,000 x g for 20-30 second centrifugation. A volume of 750 L TRIzol-prepared sample was combined with 150 L chloroform in these tubes and hand-shaken vigorously. Phase separation was accomplished by centrifugation at 1500 x g for 5 min at 4C. The aqueous phase was then mixed with 400 L ethanol 95-100% in a separate tube. These preparations were then transferred to Zymo Research RNA clean and concentrator-25 kit columns (Zymo Research, cat. R1018) and subjected to multiple wash and centrifugation steps as recommended by the manufacturer. An in-column DNase I treatment was performed using Qiagen DNase (Qiagen, 79254). Finally, RNA was eluted with 50 L DNase/RNase-Free water and stored at -80C. Sequencing: Poly-A enriched libraries were made using the TruSeq stranded mRNA LT kit (Illumina, Cat# 20020594) and sequenced on a NovaSeq SP with PE150 read length.