Lysis binding buffer

Cells were seeded into 35-mm dishes (TrueLine) at a density of 1 × 10⁶ cells/well and pre-cultured for 3 days. The cells were then collected by pipetting with culture medium into 1.5-mL tubes followed by centrifugation at 300× g for 5 min at 4 °C. The resulting cell pellets were rinsed twice with 1 mL of PBS(−) and suspended in 600 μL of lysis buffer prepared from Lysis/binding buffer (Roche Diagnostics, Mannheim, Germany) and stored at −80 °C until use for preparation of total RNA.
Total RNA was extracted from the cell lysates using a High Pure RNA Isolation Kit (Roche Diagnostics) according to the manufacturer’s instructions. The concentration and quality of total RNA in each extract were measured spectrophotometrically and evaluated by calculating the A260/A280 values, respectively (DU640; Beckman Coulter, Fullerton, CA, USA). Thereafter, the total RNA was reverse transcribed using a High-Capacity cDNA Reverse-Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and random hexamers as a primer according to the manufacturer’s instructions.

Sample inactivation and RNA extraction were performed at the MDPH, Broad Institute, and CDC. At MDPH, viral samples were inactivated by adding 300 μL Lysis/Binding Buffer (Roche) to 200 μL sample, vortexing for 15 seconds, and incubating lysate at room temperature for 30 minutes. RNA was then extracted following the standard external lysis extraction protocol from the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) using a final elution volume of 60 μL. At the Broad Institute, samples were inactivated by adding 252 μL Lysis/Binding Buffer (ThermoFisher) to 100 μL sample. RNA was then extracted following the standard protocol from the MagMAX Pathogen RNA/DNA Kit (ThermoFisher) using a final elution volume of 75 μL. At CDC, RNA extraction followed the standard protocol from the QiaAmp Viral RNA mini kit (Qiagen).

Cells were harvested from three replicates for each condition at 8 and 24 h stimulation and centrifuged 5 min at 300×g. The pellet was suspended in 200 µl PBS and lysed in 550 µl Lysis/binding buffer (Roche) supplemented with 1% DTT (Sigma Aldrich). Cell lysates were stored at − 80 °C until RNA isolation with the MagNA Pure LC RNA isolation kit-High performance (Roche) on the MagNA Pure LC 2.0 instrument following the manufacturer’s protocol. Isolated RNA samples were quantified using UV spectroscopy (Take3 module, Synergy Mx, BioTek, Winooski, USA). RNA integrity was analysed by agarose gel electrophoresis using 2% E-Gel EX pre-stained gels (Thermo Fischer Scientific).