cGAMP enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s protocol using the Cayman Chemical 2′3′-cGAMP ELISA Kit (Bertin Bioreagents). MEFTrex−/− were seeded at 3 × 106 in 150-cm2 plates 24 hours before transduction. Forty-eight hours later, cells were seeded at 3 × 106, and 24 hours later, cells were transfected with RNA:DNA hybrids (2 µg/ml). For cGAMP quantification, cells were harvested, counted, washed in phosphate-buffered saline (PBS), pelleted, and frozen in 500 μl of 80% MeOH/H2O (80/20, v/v) at −80°C until extraction. Samples were thawed and subjected to five freeze/thaw cycle in liquid nitrogen. Lysates were centrifuged at 16,000 rcf at 4°C for 20 min. The recovered supernatants were subjected to speed vacuum drying in SAVAN 110 SpeedVac Concentrator at 65°C for 2 hours. The pellets were resuspended in 50 μl of RNase/DNase-free water.

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