4sU-seq of MCMV

Swiss murine embryonic fibroblasts (NIH-3T3) were infected with wild-type Smith strain mouse cytomegalovirus (MCMV) at a multiplicity of infection (MOI) of 10. Sequencing libraries were prepared using the 4sU-seq protocol (Dölken et al., RNA 2008 and Rutkowski et al.) from the indicated time points of infection as described in Rutkowski, A.J. et al, Nat. Commun (2015) Overall design: MCMV infection was conducted on NIH-3T3 cells at an MOI of 10. Nascent RNA was labelled with 200µM 4sU one hour prior to RNA harvesting. 4sU labelled RNA was biotinylated and separated from 'old' RNA using streptavidin-coated magnetic beads (Miltenyi). Sequencing was conducted for both new and total RNA. While rRNA was depleted from total RNA, no rRNA depletion was performed for all 4sU-RNA samples. Two biological replicates were performed for each of the time points: 0,1,2,4,6,12,18,24,48 hpi