Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

Danielle E. Goodman; Carla D. Pretto; Tomas A. Krepostman; Kelly E. Carnahan; Katherine R. Spindler

RNeasy Mini Kit (Qiagen #74134) and stored at -80˚C. 125 ng of RNA per sample was used to 529 make cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems 530 #4368814), and 2 µL of the cDNA was analyzed by qPCR using a primer/probe set specific to 531 mouse PKR sequence (Thermo Fisher, Mm01235643_m1, #4331182). The results were 532 normalized to GAPDH, which was analyzed using a GAPDH-specific primer/probe set 533 Kit, Thermo Scientific #78410) was added to the plate. The cells were allowed to lyse at room 542 temperature for 10 minutes before being harvested and centrifuged at 4˚C at 14,000 x g for 10 543 minutes to remove debris. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/584680 doi: bioRxiv preprint (final concentration, Sigma C6628) was added to the media. At 24 hpi, at room temperature, 549 cells were washed once with PBS and Pierce TM RIPA lysis buffer (Thermo Scientific #89900) 550 with 1x protease inhibitors (Protease Inhibitor Cocktail Kit, Thermo Scientific #78410) was 551 added to the plate. The cells were allowed to lyse at room temperature for 10 minutes before 552 being harvested and centrifuged at 4˚C at 14,000 x g for 10 minutes to remove debris. 553