Accuscript rt

Viral RNA was isolated from plasma samples obtained after initial maximal VL suppression was achieved (<50 vRNA copies/mL), according to the previously described ultracentrifugation based vRNA extraction procedure (See above). Viral RNA was converted to cDNA using AccuScript RT (Agilent, Santa Clara CA) in a gene-specific primed cDNA synthesis reaction. The previously described reaction conditions were modified to use 21.4 µL of vRNA template, 2 µM of the gene-specific primer RT-SGA-GSP (Table S1), and omitted the initial 25°C elongation step. After cDNA synthesis, 10 U of RNAse H (NEB) and 4.4 µL 10X RNaseH buffer was added to the reaction mixture which was then incubated at 37°C for 20 min. The first round of PCR (Table S2; SGA-RT-Round 1) was performed by preparing 16, 25 µL reactions containing 2.5 µL of cDNA per reaction. A nested PCR amplification step (Table S2; SGA-RT-Round 2) was performed in a 20 µL reaction volume containing 0.5 µL of 1^st round PCR product. PCR reactions were screened by agarose gel electrophoresis. Positive amplification products were reamplified in a 50 µL reaction volume, purified, and bi-directionally sequenced using primers RT-SGASeqF and RT-SGASeqR (Table S1) by the University of Michigan's DNA sequencing core according to protocols for Applied Biosystems DNA Sequencers (Model 3730 XL).