shRNA-expressing retroviral particles were produced by cotransfection of 2 × 106 293T cells with 5 μg of shRNA-containing pSUPERIOR, 2.5 μg of murine leukemia virus (MLV) GagPol, and 2.5 μg of A-MLV envelope, using the standard calcium phosphate transfection protocol. Retroviral particles containing the transgene encoding Flag- and HA-tagged LysRS (F/HA-LysRS) were produced following the same procedure except that cells were cotransfected with 5 μg of pOZ-F/HALysRS, 2.5 μg of MLV GagPol, and 2.5 μg of A-MLV envelope. Viral particles were harvested 48 hours after transfection, filtered with 0.45 μM filters, and used for transduction.

For knockdown of LysRS, 105 cells were seeded 24 hours before transduction. Medium was replaced 10 hours after transduction, and transfection was performed 72 hours later. A similar procedure was used for knockdown of LysRS and STING, except that retroviral particles containing shRNAs targeting LysRS and STING were added at the same time. For expression of F/HA-LysRS, 5 × 105 cells were seeded 24 hours before transduction. Twenty-four hours later, viral particles were added, and medium was replaced after 10 hours. RNA:DNA hybrids were transfected 96 hours later.

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