Vargulin substrate

All HEK293FT cells were grown in DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher) in an incubator at 37°C and 5% CO₂ atmosphere. All in vitro luciferase experiments for the split-ADAR2 were carried out in HEK293FT cells seeded in 96-well plates, at 25–30% confluency, using 400 ng total plasmid and 0.6 μl of commercial transfection reagent Lipofectamine 2000 (Thermo Fisher). Specifically, every well received 100 ng each of the Cluc-W85X(TAG) reporter, N- and C-terminal ADAR2 fragments and the adRNA plasmids. In cases where less than four plasmids were needed, a balancing plasmid was added to keep the total amount per well as 400 ng. Forty-eight hours post transfections, 20 μl of supernatant from cells was added to a Costar black 96-well plate (Corning). For the readout, 50 μl of Cypridina Glow Assay buffer was mixed with 0.5 μl Vargulin substrate (Thermo Fisher) and added to the 96-well plate in the dark. The luminescence was read within 10 min on Spectramax i3x or iD3 plate readers (Molecular Devices) with the following settings: 5 s mix before read, 5 s integration time, 1 mm read height.

All HEK293FT cells were grown in DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher) in an incubator at 37°C and 5% CO₂ atmosphere. All in vitro luciferase experiments for DMS validations were carried out in HEK293FT cells seeded in 96-well plates, at 25–30% confluency, using 250 ng total plasmid and 0.5 μl of commercial transfection reagent Lipofectamine 2000 (Thermo Fisher). Specifically, every well received 100 ng of the Cluc-W85X(TAG) or Cluc-W85X(TGA) reporters, 50 ng of MCP-ADAR2-DD mutants, and 100 ng of the MS2-adRNA plasmids. In cases where less than three plasmids were needed, a balancing plasmid was added to keep the total amount per well as 250 ng. Forty-eight hours post transfections, 20 μl of supernatant from cells was added to a Costar black 96-well plate (Corning). For the readout, 50 μl of Cypridina Assay buffer was mixed with 0.5 μl Vargulin substrate (Thermo Fisher) respectively and added to the 96-well plate in the dark. The luminescence was read within 10 min on Spectramax i3x or iD3 plate readers (Molecular Devices) with the following settings: 5 s mix before read, 5 s integration time, 1 mm read height.