MLV integrations in human HepG2 cells - MiSeq run
We amplified DNA fragments containing the 3’ end of the MLV provirus using a modified version of the LM-PCR method described in http://dx.doi.org/10.1126/science.1083413.  We began with two flasks of cells; we split the DNA from each flask into thirds, each of which was digested overnight with a combination of MseI/PstI, BfaI/BanII, or Csp6I/Eco24I. Each cocktail consisted of a 6-bp cutter that prevents internal amplification by cutting downstream of the MLV 5’ long terminal repeat (LTR), and a 4-bp cutter that produces a 5’ TA overhang. We pooled digested DNA and annealed 6-bp barcoded linkers to the TA overhangs with T4 DNA ligase. We performed two rounds of nested LM-PCR. The library itself was constructed annealing paired-end adapters from the Paired-End DNA Sample Prep Kit (Illumina) to the purified amplicons from the LM-PCR.