Total RNA-Seq in HSV-1 infected human fibroblasts with spliceosome inhibition
Confluent MRC5 cells were infected with 10 PFU HSV-1 (strain KOS) per cell. 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, cell were treated with major spliceosome inhibitors at 1 or 3 hours post infection. At these times, media was removed from infected cells and replaced with media containing 30 uM Isoginkgetin (CAS #548-19-6, Sigma #416154), 25 nM Pladienolide B (CAS #445493-23-2, SantaCruz #sc-391691), or vehicle (DMSO, 0.1%). Total RNA was collected at 24 hours post infection using the Direct-zol RNA MiniPrep Kit. ERCC spike-in controls were added to total RNA. Samples 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 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.