GSM5289766: NL4.3 integration in control 293T; Homo sapiens; OTHER
Genomic  DNA was purified from cell lysis with by phenol chloroform. DNA was sheared into 300 bp-500 bp random fragments using Covaris Adaptive Focused Acoustics (Covaris, Woburn, MA). The sheared DNA fragments were end-repaired and a single dA was added to the 3' ends using KAPA DNA Hyper Prep kit . A partially double stranded linker (5'GTAATACGACTCACTATAGGGCTCCGCTTAAGGGACT3', 5'–PO4-GTCCCTTAAGCGGAG-3'-C6) with a one nucleotide 3' T overhang was ligated to the genomic DNA fragments. The 3' dA overhang on the genomic DNA fragments and the 3' T overhang on the linker DNA prevented genomic-genomic DNA and linker-linker ligation, increased the efficiency of the desired ligation, and reduced the artifacts associated with cross-ligation commonly seen with restriction enzyme based linker-mediated PCR (LM-PCR). The integration junctions were selectively amplified using one primer that matched the HIV LTR sequence and the other primer that matched the single stranded portion of the linker. Nested PCR was performed with an inner primer pair; one primer matched the sequence of the LTR and the other primer matched the linker (outer: HIV-3LTR, 5'TGTGACTCTGGTAACTAGAGATCCCTC3'; Inner: HIV-3LTRnest, 5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNCCCTTTTAGTCAGTGTGGAAAATC3'; outer: HIV-5LTR,TCAGGGAAGTAGCCTTGTGTGTGGT; inner: HIV-5LTRnest,  5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGTCTTTTTTGGGACCAAATTAGCCC; outer: Linker-P1, 5'GTAATACGACTCACTATAGGGC3'; inner: PE2_Linkernest, 5'CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNAGGGCTCCGCTTAAGGGAC3'; NNNNNN stands for barcodes).