Extraction of genomic DNA from HIV-1-infected cells.

gDNA was extracted as previously reported, with the following modifications (15, 35). Briefly, 1.25 × 10⁶ PBMC were pelleted at 500 × g for 15 min at 4°C in a 1.5-mL microcentrifuge tube, the supernatant was removed, 600 μL of lysis buffer was added, the tube was vortexed briefly for 3 to 4 s, and cells were incubated in lysis buffer for 10 min at room temperature (15). After incubation, 600 μL of 100% isopropanol was added to the tube(s), and the tube(s) was inverted >30 times for sufficient mixing (to minimize gDNA shearing) and centrifuged at 21,100 × g for 20 min at 10°C, after which the supernatant was removed. The pellet was washed by the addition of 1 mL of ice-cold 70% ethanol and mixed by inverting the tube >30 times, the contents were centrifuged at 21,100 × g for 20 min at 10°C, the supernatant was carefully removed, and the ethanol wash was repeated twice more. The gDNA pellet was air dried for 15 min at room temperature and resuspended in 200 μL of ice-cold 5 mM Tris-HCl (pH 8.0) by careful pipette mixing of the DNA.