title: Integration sites from HIV-Luc-infected U2OS siLEDGF 21 cells
design: Sowd et al. (PNAS. 2016) infected siLEDGF L21 U2OS cells (LEDGF/p75 knockdown) with VSVg-pseudotyped HIV-Luc at 1.67 pg p24 HIV-Luc/cell.  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.