Analysis of CSR Junction Mutations

B cells were isolated from mouse spleens (n = 6 per genotype) and stimulated for class-switching in culture for 72 hr. Where indicated, cultures were incubated with DNA-PKcs inhibitor 20 µM NU7026 (Tocris, Bristol, UK) dissolved in DMSO, or mock-treated. The stimulation procedure and flow-sorting for CSR analysis was as described [31] (link), [67] (link). Prior to this analysis, cells were counted; numbers and viability were similar for all groups. Sμ-Sγ1 CSR junctions were amplified by PCR using the following conditions for 25 cycles at 95°C (30 s), 55°C (30 s), 68°C (180 s) using the primers (FWD 5′-AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3′; REV 5′ CAATTAGCTCCTGCTCTTCTGTGG-3′) and Pfu Turbo (Stratagene, La Jolla, CA). To the PCR reaction, 5 U of Taq polymerase (Promega, Madison, WI) was added and incubated at 72°C for 10 min. The resulting product was TOPO TA cloned and transformed into Top10 E. coli cells (Life Technologies, Carlsbad, CA) and plasmids were purified and sent for sequencing using M13 FWD and REV primers in addition to the amplification primers for sequencing. 100 clones for each group were analyzed for mutations, deletions, insertions, and sequence overlaps at the junction and both 30 nt upstream and downstream of the junction. p-values were determined by using two-tailed Fisher's exact test.