Comparison of HCMV nascent and steady state transcriptomes expresssed in the Kasumi-3 latency model at early times post-infection

Purpose: to compare newly transcribed and steady-state HCMV RNAs expressed in Kasumi-3 cells at 24 hr post-infection Overall design: Kasumi-3 cells were infected with the TB40/E wtGFP strain of HCMV. At 24 hr post-infection, cells were sorted for GFP expression, and newly synthesized RNA was labeled for 4 hr with 5-ethynyl uridine. Mock-infected cells were sorted for live cells and labeled in parallel. A portion of the RNA was save for analysis of steady state RNA, and the nascent RNA was purified by biotinylation of EU-labeled RNA and selection on Streptavidin beads. cDNA was prepared from each of the four RNA populations and the cDNA was purified with a Qiagen minElute PCR purification kit. Nextera libraries were prepared and sequenced with Illumina NextSeq paired end technology.