Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA from different virus strains and mutant viruses

Primary human foreskin fibroblasts (HFF) were infected with the indicated HSV-1 strains and mutants thereof at a multiplicity of infection (MOI) of 10. Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013. Overall design: Newly transcribed RNA was labelled for one hour at the indicated times of infection. Cells were harvested immediately thereafter using Trizol. Following isolation of total RNA and thiol-specific biotinylation, 4sU-RNA was purified and subjected to RNA-seq following rRNA depletion using Ribo-zero. Two independent biological replicates were analysed.