Herpesvirus infection reduces Pol II occupancy of host promoters but spares viral promoters

Ella N Hartenian; Britt A Glaunsinger

The WT and R443I MHV68 bacterial artificial chromosome (BAC) were previously 361 described (6,50). MHV68 was produced by first making p0 virus by transfecting NIH 3T3 cells 362 in 6-well plates with 2.5 µg BAC DNA using TransIT-X2 (Mirus Bio) for 24h. Five to seven 363 days later, the cells were split into a 10 cm dish, then harvested and frozen. After 5-7 days the 364 majority of cells showed cytopathic effect (CPE). 30 µL of p0 was then added per confluent 10 365 cm dish of NIH 3T12 cells, split 2 days later into four 10 cm dishes and harvested 4-6 days later 366 when all cells were infected. Cells were collected by centrifugation (5 min, 1500 rpm) and the 367 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/585984 doi: bioRxiv preprint pellet was dounced 10 times. The cell pellet and supernatant were then ultracentrifuged for 2 h at 368 30,000 rpm. The resulting pellet was resuspended and titered by plaque assay. Cells were 369 infected with MHV68 at an MOI of 5 for 24 hours. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/585984 doi: bioRxiv preprint custom index was made of the mm10 and MHV68 genomes with Bowtie2 build. Reads were 437 mapped to that index using Bowtie2 (2.3.0). 438