One million HIV-1 insertion sites in HEK293
HEK293 and HOS cells were transfected with 0.5-1ug VSVg pseudotyped pNLNgoMIVR-Emodluc and genomic DNA was isolated ~4 days following infection. DNA was digested with the MseI restriction enzyme, fragments were purified, and ligated with an MseI-cut specific adapter. Linker-ligated fragments were digested with the BglII restriction enzyme before being amplified by PCR with linker-specific and HIV LTR-specific primers. PCR products ranging from 200bp to 500bp were purified and loaded onto 6 lanes of an HiSeq2000 for Illumina pair-end sequencing. The sequencing barcodes were used as follow: - lanes P1 and P2: barcodes ACGTC and CTAGT for HEK293 cells; barcodes GATCA and TGCAG for HOS cells - lanes K1 and K2: barcodes ACGTC, GATCA, CTAGT, TGCAG, TAGC and ATCG for HEK293 cells - lanes S1 and S2: barcodes ACGTC, GATCA, TAGC and ATCG for HEK293 cells; barcodes CTAGT and TGCAG for HOS cells