SARS-CoV-2 RNA FISH Protocol

RNA FISH was performed following a previously published protocol^{68 (link)}. Briefly, a set of FISH probes targeting 229E viral RNA was designed and ordered from IDT and labeled with AF674 (Thermo Scientific) following our previous work^{69 (link)}. The cells were cultured in 8-well μ-slides (ibidi Cat# 80824). Cells were washed with PBS, fixed with 3.7% formaldehyde in PBS at room temperature for 10 min, washed twice with PBS, and then permeabilized with 70% (vol/vol) ethanol for >1 h at 4°C. After decanting 70% ethanol from the wells, 200 μL of Wash Buffer A (40 μL Stellaris® RNA FISH Wash Buffer A (Cat# SMF-WA1–60, LGC Biosearch Technologies), 20 μL of deionized formamide, 140 μL distilled nuclease-free H₂O) was added to cells and incubated at room temperature for 5 min. After decanting Wash Buffer A, 100 μL of Hybridization Buffer (90 μL Stellaris^® RNA FISH Hybridization Buffer (Cat# SMF-HB1–10, LGC Biosearch Technologies), 10 μL of deionized formamide) containing 2 μL of 12.5 μM RNA FISH probes was added into each well and incubated for 16 h at 37 °C in the dark. Then cells were washed twice with Wash Buffer A by incubating for 30 min at 37°C in the dark and stored in Wash Buffer B (Cat# SMF-WA1–60, LGC Biosearch Technologies) for imaging.