LM-PCR to amplify MLV integration sites of mice spleen infected with WT MLV (WT10)
Genomic DNA sample (5 g) was subjected to two rounds of sonication with the following parameters: duty cycle: 5%; intensity: 3; cycles per burst: 200; time: 80 sec). Purification of DNA for the next generation library protocol used MinElute Reaction Cleanup kit (Qiagen 28204). The sonicated DNA was purified and ends of DNA fragments were repaired using End-It DNA End-Repair Kit (ER0720) as described in manufacturers protocol and purified after repaired DNA ends were A-tailed using Klenow Fragment (M0212S). All kits were used as described by the manufacturers protocol. Linker short and long strands (S6 Table) were annealed by heating to 90 C and slowly cooling to room temperature in steps of 1 C per min. The annealed linkers were ligated with assigned genomic DNA sample with 3000 U of T4 ligase (M0202M) overnight at 12 C and purified. Semi-nested PCR using LTR specific primers and linker specific primer was used to amplify integration sites.
SUB5844609
Gim16(WT) inf-10