Integration sites from HIV-GFP-infected monocyte derived macrophage siLEDGF L3 cells
Prior to infection, siLEDGF L3 monocyte derived macrophages (MDM) (LEDGF/p75 knockdown) were pretreated with virus-like particles containing the simian immunodeficiency virus Vpx protein. Sowd et al. (PNAS. 2016) infected siLEDGF L3 MDMs with VSVg-pseudotyped HIV-GFP at an approximate MOI of 0.6.  DNA was extracted from 2 to 5 million cells using the DNeasy Blood and Tissue Kit (Qiagen). Ten micrograms of DNA was digested with MseI and BglI, ligated to adapters, and amplified by ligation-mediated PCR as described by Matreyek et al. Retrovirology (2014).  A second round of nested PCR was performed to further amplify HIV-1 LTR containing PCR fragments. Nested PCR primers contained unique linker sequences.  PCR products were sequenced using the Illumina MiSeq platform and single-end 150 bp next-generation sequencing.