Schematic representation of the genome-wide shRNA screening workflow. HeLa CD4++ cells were transduced with a pooled shRNA lentiviral library. Productively transduced cells were selected with puromycin and transduced with a retroviral vector for the expression of HIV-1 Nef and eGFP as a reporter gene. Cells that maintained high CD4 surface levels despite the expression of Nef (Nef(eGFP)+CD4hi, red box) and the Nef(eGFP)+CD4low population (green box) were sorted by FACS. The shRNA hairpins present in the cDNA obtained from the two sorted populations were amplified by nested PCR with biotinylated primers. The samples obtained were hybridized on different Affymetrix U133 v2-plus microarrays and the results compared. shRNA sequences enriched in the Nef(eGFP)+CD4hi cells were selected and further analyzed with pathway analysis tools to obtain a final list of 55 genes to be further validated together with 20 genes selected from literature.