Innate Immune Priming by cGAS as a Preparatory Countermeasure Against RNA Virus Infection

Michael T. Parker; Smita Gopinath; Corey E. Perez; Melissa M. Linehan; Jason M. Crawford; Akiko Iwasaki; Brett D. Lindenbach

To detect cGAMP by liquid chromatography and mass spectrophotometry, 554 trypsinized cells were washed once with DPBS, pelleted at 1,000 x g and then frozen 555 at -20°C. To extract cGAMP, 5x10 6 cells/mL were resuspended three times in 556 extraction buffer (40% acetonitrile, 40% methanol, 20% ddH 2 O) for 20 minutes, 557 spinning after each extraction at 16,100 x g and keeping the supernatant. 558 and incubated for 10 minutes at 4°C with rotation. A succession of 4°C spins was 573 performed, retaining the supernatant for each step: 3x 1,000 x g for 3 minutes, 1x 574 16,100 x g for 10 minutes, 1x 100,000 x g for 1 hour on a 0.34 M sucrose cushion 575 (SW41 Ti rotor, Beckman). The final supernatant was then processed for western 576 blotting, above, and DNA purification, below. 577 DNA purification and phi29 amplification. DNA was isolated from total cytosol 578 by treating samples with RNase A and RNase T1 (Ambion) for 1 hour at 37°C, 579 digesting with Proteinase K for 1 hour at 55°C, and heat inactivating at 95°C for 15 580 minutes. DNAs were then purified with the QiaQuick purification kit (Qiagen). To 581 isolate DNA from immunoprecipitates, crosslinks were reversed by adding 5 M NaCl 582 The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/434027 doi: bioRxiv preprint