Tiled Array Experiments Determining RNA Expression in gHV68 infected 3T12 cells

Viral genetic studies often focus on large open reading frames (ORFs) identified during genome annotation (ORF-based annotation). Here we provide a tool and software set for defining gene expression by murine gammaherpesvirus 68 (γHV68) nucleotide-by-nucleotide across the 119,450 basepair (bp) genome. These tools allowed us to determine that viral RNA expression was significantly more complex than predicted from ORF-based annotation, including over 73,000 nucleotides of unexpected transcription within 30 expressed genomic regions (EGRs). Approximately 90% of this RNA expression was antisense to genomic regions containing known large ORFs. We verified the existence of novel transcripts in three EGRs using standard methods to validate the approach and determined which parts of the transcriptome depend on protein or viral DNA synthesis. This redefines the genetic map of γHV68, indicates that herpesviruses contain significantly more genetic complexity than predicted from ORF-based genome annotations, and provides new tools and approaches for viral genetic studies. Study includes a total of 12 samples. These are 3 replicates of gHV68-infected 3T12 cells 18hpi, 3 replicates of gHV68-infected 3T12 cells 8hpi, 3 replicates of gHV68-infected 3T12 cells 8hpi treated with cyclohexamide, and 3 replicates of gHV68-infected 3T12 cells 18hpi treated with cidofovir. Each of these samples has a matching mock-infected 3T12 sample which was subtracted to remove host RNA crosshybridization and are attached as extra raw data files.