Isoamyl alcohol

MH-S cells were carefully washed with pre-chilled PBS 2 times. Pre-chilled RIPA lysis buffer (50 mM PH7.4 Tri-HCl, 15mM NaCl, 1mM EDTA, 0.1% NP-40, 1.25% Triton X-100, protease inhibitor) was added, and then MH-S cells were collected to sterilized 1.5 ml Eppendorf tubes using a pre-chilled scraper, then cells were centrifuged at 14,000 g/min for 15 min at 4 °C, the supernatant was incubated with the magnetic agarose beads containing the antibody overnight (Invitrogen, Dynabeads M-280 sheep anti-rabbit). Cells were washed twice with low salt wash buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40), and then were divided into two equal parts, one for detecting IP efficiency by western blotting. Another magnetic bead sample was added with 200 μl of DNA decrosslinking solution (10 mM Tris, 1Mm EDTA, 0.65% SDS), incubated at 65 °C for 16 h, and then proteinase K (20 mg/ml) was used to digest protein by incubating for 2 h at 37 °C. The DNA that bound to the cGAS protein was extracted through the mixture of phenol, chloroform, and isoamyl alcohol (25:24:1) (Thermo Fisher), and then was precipitated in 75% ethanol. Finally, the DNA was dissolved in the nuclease-free water and quantified by qPCR by using: Cytochrome c oxidase I (mtDNA gene) and housekeeping gene (18S rDNA) (27 (link)), the PCR primers used in the reaction are listed in Supplementary Table S1.