Figure 1

Strategy of cDNA library generation with hybridization capture for HIV-1 encoded small noncoding RNAs (sncRNAs). HIV-1 susceptible cells (in our set-up, primary human macrophages or CD4⁺ T-lymphocytes) are infected with HIV-1 (Step 1). Cellular (black) and HIV-1 encoded (bright green) sncRNAs (< 200 nt) are extracted from HIV-1 infected cells (Step 2). RNA is C-tailed at the 3'-end, adaptor-ligated at the 5'-end (Step 3), and RT-PCR is performed (Step 4). For the preparation of the HIV-1 ssDNA hybridization probes, PCR is performed with biotinylated primers for 5 overlapping regions of the genome using HIV-1_JR-FL plasmid as template. Biotinylated amplicons are attached to streptavidin beads (Box 1). Sequences homologous to HIV-1 are enriched by incubation of cDNA derived from adaptor-ligated sncRNAs with a mixture of the 5 different HIV-1 ssDNA hybridization probes (Step 5); alternatively each HIV-1 ssDNA hybridization probe can be used separately. After hybridization capture, bound amplicons are eluted, amplified, and size selected on a gel (Step 6). The hybridization and size selection steps can be repeated (Step 7). Amplicons are cloned and sequenced or can be sequenced using next-generation sequencing technologies (Step 8).