title: Total RNA-Seq in KSHV Infection Models
design: HUVEC (HUV) or HDLEC (LEC) cells were infected with KSHV strain BAC16 at an approximate MOI of 0.25 (30% cells infected) or 1 (70% cells infected), respectively. Virus was adsorbed in a low volume of media for 8 hr at 37 degrees C, after which viral inoculum was removed and replaced with fresh media. 0 hour time point was when virus was added and cells were first placed at 37 degrees C to incubate. Total RNA was isolated at 3 days post infection using the Direct-zol RNA MiniPrep Kit. For lytic reactivation models, subconfluent monolayers of iSLK-BAC16 (WT, deltaORF24, deltaORF57) were induced with 1 ug/mL Doxycycline, 1 mM Sodium Butyrate in 1x DMEM media supplemented with 2% Tet-approved FBS. 0 hour time point was when induction media was added and cells were first placed at 37 degrees C to incubate. If indicated, cells were treated with 100 uM Cidofovir or 70 ug/mL Phosphonacetic Acid at 0 hr post induction. Total RNA was isolated at 24 or 72 hours post induction using the Direct-zol RNA MiniPrep Kit. RNA samples from LEC infected with BAC16 for 3 days were also subjected to RNaseR digestion followed by sequencing. For these samples, 3 ug total RNA was combined with 20 Units RNase Inhibitor, 20 Units RNase R, 100 mM KCl, 20 mM Tris-HCl pH 8.0, 0.1 mM MgCl2. RNA was RNase R digested at 37 degrees C for 30 minutes, following clean-up with the RNA Clean and Concentration Kit. For Total RNA (no RNaseR digestion) ERCC spike-in controls were added. RNA was ribominus selected and directional cDNA libraries were generated using either Stranded Total RNA Prep with Ribo-Zero Plus (Illumina # 20040525) or TruSeq Stranded Total RNA Ribo-Zero Gold (Illumina #RS-122-2303). 2-4 biological replicates were sequenced for all samples. Sequencing was performed at the NCI CCR Frederick Sequencing Facility using the Illumina NextSeq 550 or Illumina NovaSeq SP platform to generate 150 bp PE reads.