Immunofluorescence Staining Protocol

Cell fixation and antibody staining were performed as previously described^{7 (link)}. Briefly, cells were fixed with ice-cold (−30 C) methanol for 15 minutes – when staining for centromeres, centrosomes, cGAS, Vimentin, β-actin, IRF3, or a-tubulin – or 4% paraformaldehyde – when staining for RelB, p65, STING, ssDNA, dsDNA, CoxIV, or β-catenin. Subsequently, cells were permeabilized using 1% triton for 4 minutes. See supplementary Table 1 for antibody information. For selective plasma membrane permeabilization used for cytosolic dsDNA and ssDNA staining, cells were treated with 0.02% saponin for 5 minutes after fixation. For single-stranded (Thermo Fisher FEREN0321) and double stranded (Life Technologies – EN0771)-specific nuclease treatment, cells were also permeabilized with 0.02% saponin for 2 minutes and treated with either nucleases for 10 minutes before fixation using 4% paraformaldehyde. TBS-BSA was used as a blocking agent during antibody staining. DAPI was added together with secondary antibodies. Cells were mounted with Prolong Diamond Antifade Mountant (Life Technologies – P36961). cGAMP (Invivogen – tlrl-nacga23) was transfected into cells at a concentration of 4μg/ml using lipofectamine2000 that was added for 3–4 hours and then replaced with regular serum containing medium.