In vitro interaction between RNA:DNA hybrids and recombinant proteins was performed using Dynabeads M280. Beads were blocked overnight in blocking buffer [20 mM Hepes (pH 7.9), 15% glycerol, 0.05% NP-40, and 50 mM NaCl, supplemented with 2 mM dithiothreitol (DTT), 100 mM NaCl, and bovine serum albumin (BSA; 10 mg/ml)]. After three washes in 1× wash buffer [5 mM tris-HCl (pH 7.5), 1 mM EDTA, and 1 M NaCl], M280 beads were coupled to nucleic acids according to the manufacturer’s instructions. Briefly, beads were incubated in 30 μl of 2× wash buffer [10 mM tris-HCl (pH 7.5), 2 mM EDTA, and 2 M NaCl] containing 20 pmol of BRNA:DNA hybrids at 25°C for 15 min. Protein binding was performed at 37°C for 30 min with increasing amounts of recombinant proteins in 20 mM Hepes (pH 7.9), 15% glycerol, 0.05% NP-40, and 150 mM NaCl, supplemented with 2 mM DTT, 2 mM PMSF, and RNase inhibitor. When indicated, hybrids were treated with 30 U of RNaseH (Ambion) in RNaseH buffer [50 mM tris-HCl (pH 8), BSA (50 ng/ml), 1 mM DTT, and 4 mM MgCl2] for 1 hour at 30°C, before binding to the beads. After three washes, bound material was eluted in 30 μl of Laemmli buffer. Pull-down in competition between hcGAS and hLysRS was performed as described above except that the two proteins were added at the same time to the beads coupled to the BRNA:DNA hybrids.

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