Fragile X Mental Retardation Protein Regulates R-loop Formation and Prevents Global Chromosome Fragility

We adapted a previously published method, Break-seq, which was developed in the model organism Saccharomyces cerevisiae to mammalian cells and mapped DNA double strand breaks (DSBs) genome-wide. EBV transformed lymphocytes derived from a normal individual(GM06990) and a Fragile X individual (GM03200) were grown in RPMI1640, supplemented with GlutaMAX, 15% heat-inactivated FBS, 100 IU/ml penicillin and 100 ug/ml streptomycin. At a density of 0.4-0.5x10E6 cells/ml, cells were treated with solvent (DMSO, 0.02%), 0.03 micromolar aphidicolin (APH), 0.3 micromolar APH, or nothing at all. APH was dissolved in 0.02% DMSO at both concentrations. After 24 h treatment, cells were harvested for Break-seq analysis. Three independent sets of experiments were performed, wherein Set A and B are technical replicates from the same experiment and Set D and E are biological replicates. Each strain/treatment combination has at least two indepent experiments.