Integration_site_analysis:B+R+
Timer populations from bulk-infected Jurkat cells with the HIVTimer molecular construct were sorted and genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturers recommendations. About 2 g of genomic DNA was sheared by sonication with a Picoruptor (Diagenode, S.A., Belgium) instrument to a size of 300400 bp in length. DNA end was repaired and the addition of adenosine at the 3 end of the DNA was performed with NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Cat# E7546). A linker was ligated to the ends of DNA using the NEBNext Ultra II Ligation Module (New England Biolabs, Cat# E7595). The junction between the 3LTR of HIV-1 and host genomic DNA was amplified using primers targeting the 3LTR and linker regions. The first PCR amplicons were purified using a QIAquick PCR purification kit (QIAGEN), and then the second PCR was performed. The following thermal cycler conditions were used for both PCRs: 96 C for 30 s (1 cycle); 94 C for 5 s, 72 C for 1 min (7 cycles); 94 C for 5 s, 68 C for 1 min (13 cycles); 68 C 9 min (1 cycle) and hold at 4 C. The second PCR amplicons were purified using a QIAquick PCR Purification Kit (QIAGEN), followed by Axy Prep Mag PCR cleanup bead purification (AXYGEN, Cat# MAG-PCR-CL-50). Purified PCR amplicons were quantified using the Agilent 4150 TapeStation system, and quantitative PCR was performed using primers P5 and P7 (GenNext NGS library quantification kit, Toyobo, Code# NLQ-101). LM-PCR libraries were sequenced using Illumina MiSeq paired-end reads, and the resulting FASTQ files were used for downstream analysis.