Nuclear cytoplasmic fractionation

T98G^F/H-cGAS cells were fractionated according to Gentili et al. (26) Briefly, cells were harvested and cell pellet was washed once in 400 μl of cold cytoplasmic lysis (CL) buffer [10 mM Hepes (pH 7.9), 10 mM KCl, and 1.5 mM MgCl₂] in the presence of 0.5 mM PMSF and centrifuged at 300g for 4 min. The supernatant was discarded, and cell pellet was lysed by adding three cell pellet volumes of cold CL for 10 min on ice, with gentle flicking. Cytosolic extract was obtained by adding 0.625% final of NP-40 for an additional of 5 min on ice, with gentle flicking. Nuclei were pelleted at 16,000g for 5 min, and cytosolic extracts were collected in a fresh tube. Nuclei were then lysed by adding three cell pellet volumes of cold nuclear lysis buffer [420 mM NaCl, 20 mM Hepes (pH 7.9), 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, and 0.5 mM PMSF] for 15 min on ice, with gentle flicking. Nuclear lysates were vortexed and sonicated for 20 min at 4°C in a Bioruptor ultrasonic bath. Nuclear lysates were cleared by centrifugation at 16000g for 5 min.