Project: mRNA-seq on IFN-treated Calu-3 and Huh-7.5 cells; Cells: Huh-7.5; Treatment: Mock; Analysed data: See Manuscript Supp Tables 4 and 5
Cell culture and treatment: A549 cells: 300,000 cells were seeded in each well of a 6-well plate with 3 mL of DMEM supplemented with 10% FBS and 1% NEAA. The following day, the media was aspirated and replaced with 2 mL of DMEM with or without 0.5 nM IFN-2a (PBL, cat. 11101-2), and the cells were incubated at 37C. After 24 hours, the cells were harvested in 500 L TRIzol. N = 3 replicates (separate wells treated on the same day) per sample. Calu-3 cells: 200,000 cells were seeded in each well of a 12-well plate with 1 mL media. Two days later, the media was replaced with EMEM (ATCC, cat. 30-2003) with 10% FBS with 0.5 IFN-2a (PBL, cat. 11101-2) and incubated at 37oC. 24 h later, cells were harvested in 500 L TRIzol. N = 3 replicates (separate wells treated on the same day) per sample. Huh-7.5 cells: 75,500 cells were seeded in each well of a 12-well plate with 1 mL media. Two days later, the media was replaced with 1 mL of DMEM with 5%FBS, 1% NEAA with 0.5 nM IFN-2a (PBL, cat. 11101-2) and incubated at 37oC. 24 h later, cells were harvested in 500 L TRIzol. N = 3 replicates (separate wells treated on the same day) 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 NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, cat. E7770) and sequenced on a NovaSeq SP with PE150 read length.