HSV-1 genome sequencing in cell cultures

HEp-2/delta-hUNGs cells in 6-well plates were transfected with HSV-1 infectious genome clones of vUNG-S302A, vUNG-SA-repair in combination with pcDNA-EGFP-P2A-hAPOBEC1, pcDNA-EGFP-P2A, and pFlag-CMV2 (Sigma). Six days post-transfection, the cells were lysed in lysis buffer and proteinase K from a GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) and treated with RNase A following the manufacturer's instructions. After brief sonication, DNA was extracted twice with phenol-chloroform and four times with diethyl ether. The genomic DNA was then purified using NucleoSpin Gel and PCR Clean-up Columns. Following amplification of specific regions of the HSV-1 genome, next-generation sequencing analysis was performed.