A77V CA mutant HIV-1 integration in CD4+T cells
Achuthan et al. infected DMSO treated CD4+T cells from DonorB, after stimulation for 48 h with 5  g/ml phytohemagglutinin, with 10 pg p24 A77V CA mutant nl43-GFP/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 paired-end 150 bp next-generation sequencing.