Ta cloning

PCR products were amplified from extracellular virion DNAs using Easy-A DNA polymerase (Stratagene) and purified using QiaQuick or MinElute PCR purification kits (Qiagen), then either sequenced directly or cloned into plasmids using T/A cloning (Promega) or TOPO XL (Invitrogen) PCR cloning kits. Sanger chain termination sequencing was conducted by either the Biopolymer Laboratory at the University of Maryland at Baltimore or the Nucleic Acids Research Facility at Virginia Commonwealth University. The deletion and the GP89 mutation were confirmed by direct sequencing of R-75 and wild type GPCMV virion DNA.

Genomic DNA was isolated from 2x10⁶ cells using an E.N.Z.A Tissue DNA Kit as directed by the manufacturer (Omega Bio-tek, Norcross, GA). The isolated DNA was quantified using a NanoDrop 2000/c Spectrophotometer (Thermo fisher Scientific, Waltham, MA) and then stored at −80°C. The integration sites in each cell line were mapped based on a protocol previously described (33 (link)), but modified for Sanger sequencing by using altered primers without adapter sequences: Integration site linker (5’-GTAATACGACTCACTATAGGGC-3’), first round HIV primer (5’-TGTGACTCTGGTAACTAGAGATCCCTC-3’), and second round LTR primer (5’-GAGATCCCTCAGACCCTTTTAGTCAG-3’). Final PCR products were ligated into the pGEM-T cloning vector by TA cloning as directed by the manufacturer (Promega, Madison, WI). Clones were sequenced using the SP6 universal primer (GeneWiz, South Plainfield, NJ). Sequences were analyzed using SeqBuilder v14 software (DNAStar, Madison, WI), and the location of flanking genomic DNA in the human genome was determined using UCSC BLAT genome browser (University of California Santa Cruz). The determination of whether the location was present in a gene was determined by BLASTn search using the NCBI website.