For validation of rearrangements, we designed nested PCRs surrounding the breakpoints determined by the BRASS algorithm (primer sequences available upon request). PCR reactions were carried out in 20 μl, using 10 ng (HSC clones or tails) or 400 ng (donor bone marrow) of genomic DNA and GoTaq G2 Hot Start Polymerase (M7401, Promega). The first round of PCR amplifications was performed at 95 °C for 2 min, 35 cycles of 95 °C for 30 s, 55 °C for 20 s and 72 °C for 30 s, then 72 °C for 5 min. The reactions were diluted to 1 in 50 and 1 μl of the diluted reaction was used as template for a second round of PCR with nested primers, to increase specificity and sensitivity, performed at 95 °C for 2 min, 35 cycles of 95 °C for 30 s, 60 °C for 20 s and 72 °C for 30 s, then 72 °C for 5 min. The reactions were analysed on 2% agarose gels, bands of the expected sizes were excised and the identities of all products were confirmed by Sanger sequencing.
