Nucleotrap kit

Plasmid DNA was extracted from E. coli with the GenElute^TMHP Plasmid miniprep purification kit, as recommended by the manufacturer (Sigma). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs and Promega, respectively). Oligonucleotide primers were synthesized by Eurogentec (Seraing, Belgium). PCR was performed in a T100 thermal cycler (Biorad) with the iProof high-fidelity DNA polymerase (Biorad). Amplified DNA fragments were purified with a PCR purification kit (Roche) and separated by electrophoresis in 1% agarose gels after digestion. Hydrolyzed DNA fragments were extracted from agarose gels with the NucleoTrap kit from Macherey-Nagel. All constructs were confirmed by DNA sequencing (MWG).

Selected PCR amplicons were gel purified using the Nucleotrap kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. Purified amplicons were Sanger sequenced with integration site-specific primers. Nucleotide sequences were verified and assembled using Sequencher 4.10.1 (Gene Codes Corp., Ann Arbor, MI, USA), Geneious Prime 2020.1 (Biomatters Ltd., Auckland, NZ) and BioEdit Sequence Alignment Editor (V7.2.5). Nucleotide sequences were identified as HIV by BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch& BLAST_SPEC = OGP__9606__9558&LINK_LOC = blasttab&LAST_PAGE = blastn). Proviral sequences were identified using sequences for the human genome that were specific to each integration site and confirmed by Human BLAT Search (https://genome.ucsc.edu/cgi-bin/hgBlat). Proviral sequences were reconstructed with sequence data obtained from the Sanger sequencing reactions and the closest HIV sequence match in GenBank (AF321523).