Total RNA-Seq in HSV-1 infected human fibroblasts with RNA ligase depletion
MRC5 cells were transfected with siRNAs pools targeting human RNA ligases (RLIG1, RTCB) or a nontargeting control (NTC). At 48 hours post transfection, 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. Total RNA was collected at 12 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.