046_HIV_in_vitro_integration
The plasmid pK184 was used as an integration target for the full-length viral cDNA produced in the Endogenous Reverse Transcription (ERT) reaction. Purified reaction products were amplified by PCR using Phusion Polymerase (NEB), diluted 100-fold and measured by quantitative PCR (qPCR). Briefly, 5 l of purified ERT product was combined with 10 l Phusion HF Buffer, 200 M each dNTP, 0.24 M each of three primers (pK184-IN-FWD, pk184-IN-REV, and DC3014), 0.5 l Phusion Polymerase, and water to a final volume of 50 l. PCR cycles were as follows: Denaturation at 95C for 5 min; 23 cycles of 95C for 30 sec, 58C for 30 sec, and 72C for 90 sec; and a final extension at 72C for 5 min followed by a hold at 4C. Final PCR products were purified with a Qiaquick PCR purification kit (Qiagen) according to the manufacturers instructions and eluted in 120 l elution buffer and diluted 100-fold for qPCR analysis of 3 integration. To obtain 100 ng DNA for deep sequencing, 8 (for experimental repetitions 046 and 048) or 24 (for 047) identical PCR reactions were performed, combined, purified with a Qiaquick PCR purification kit (Qiagen), eluted in 50 l elution buffer and submitted for deep sequencing analysis. Experimental repetitions 046 and 047 used 23 PCR amplification cycles, whereas 048 was amplified for 35 cycles. These samples were analyzed by ion torrent sequencing to identify 3 integration target site sequences.