IFN Treatment of Human Epithelial Cells

All IFNs used are listed in Table S5. The IFN-treated NHBE cells were previously described^{22 (link)}. Briefly, the cells were cultured in BEGM with supplements (Lonza), plated in 6-well plates and utilized at ~70% confluence (typically after 10–11 days with media change every 2 days). Cells were left untreated or treated with IFN-α2b (INTRON A, Merck, 100 IU/ml) or IFN-λ3 (R&D Systems, 100ng/ml) for 24 hrs. Cells were washed with PBS, resuspended in TRIzol (Thermo Fisher) and stored at −80°C for future RNA isolation. Total RNA was extracted with Direct-zol mini RNA isolation kits (Zymo Research). The IFN treatment of human colon and ileum organoids was previously described²³ . Briefly, cells were seeded 24 hrs prior to IFN treatment to reach a confluency of 70% at the time of treatment. Media was removed from cells and replaced with a cocktail of IFN-λ1–3 (100ng/mL of each for a total of 300ng/mL). Media was removed 24 hrs post-treatment, and RNA was extracted using the RNAeasy kit (Qiagen). 250ng of RNA was used to prepare cDNA using iSCRIPT (BioRad) and qRT-PCR was performed using iTaq SYBR Green (BioRad). T84 and Caco-2 cells were treated with IFN-γ (2 ng/ml) for 48 hrs, followed by cell harvesting, RNA extraction and expression analysis.