Total RNA-Seq in HSV-1 infected mouse trigeminal ganglia

Female 8-week-old BALB/cAnTac mice were infected with 105 PFU HSV-1 (strain 17) via the ocular route. Latently infected trigeminal ganglion were harvested 30 days after the clearance of the primary infection. Control (uninfected) and infected trigeminal ganglion were taken from age-matched mice. Pools of 6 ganglia were homogenized in 1 ml TriPure Isolation Reagent (Roche) using lysing matrix D on a FastPrep 24 instrument (3 cycles of 40 seconds at 6 m/s). 0.2 ml chloroform was added for phase separation using Phase Lock Gel Heavy tubes and RNA isolation from the aqueous phase was obtained by using ISOLATE II RNA Mini Kit (Bioline). 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). 2-4 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.