Total RNA-Seq in HSV-1 infected human fibroblasts

Confluent MRC5 cells were infected with 10 PFU virus per cell. Viruses used in this study include: KOS (wildtype), n12 (delta ICP4), 5dl1.2 (delta ICP27), n199 (delta ICP22), and hp66 (delta UL30). Virus was adsorbed in PBS for 1 hr at room temperature. Viral inoculum was removed, and cells were washed quickly with PBS before adding on 1x DMEM media supplemented with 2% FBS. 0 hour time point was considered after adsorption of infected monolayers when cells were place at 37 degrees Celsius to incubate. If indicated, cells were treated with 75 ug/mL Cycloheximide or 300 ug/mL Phosphonacetic Acid at 0 hr post infection. Total RNA was isolated at 12 hours post infection from cells using the Direct-zol RNA MiniPrep Kit.Samples infected with KOS for 12 hours were also subjected to RNaseR digestion followed by sequencing. For these samples, 7 ug total RNA was combined with 1x E-PAP Buffer, 2.5 mM MnCl2, 1 mM ATP, 40 Units RNase Inhibitor, and 8 Units E-PAP. Poly-A tailing reactions were incubated at 37 degrees C for 10 minutes. RNA was acid-phenol chloroform extracted, ethanol precipitated, and resuspended in nuclease-free water. PolyA-Tailed material was combined with 20 Units RNase Inhibitor , 20 Units RNase R, 100 mM LiCl, 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.ERCC spike-in controls were added to total RNA. All samples (untreated, or RNaseR digested) were 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 NovaSeq SP platform to generate 150 bp PE reads.