GM12982_R1_s
The cell lines from Bloom Syndrome and control patients were obtained from the Cell Repository of Coriell Institute: GM07492 and GM07545 (primary fibroblasts, control), GM02085 and GM03402 (primary fibroblasts, BS), GM12892 (EBV-transformed B-lymphocytes, control), GM16375 and GM17361 (EBV-transformed B-lymphocytes, BS). Mouse ES cell line F121.6 (F1 hybrid between 129/Sv and Cast/EiJ) was a kind gift from Prof. J. Gribnau (Erasmus MC, Rotterdam, The Netherlands). Blm mutant ES cell lines were generated as previously described (7). Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% v/v fetal bovine serum (FBS) (Sigma Aldrich) and 1% v/v penicillin-streptomycin (Life Technologies), B-lymphocytes in RPMI1640 (Life Technologies) supplemented with 15% v/v FBS and 1% v/v penicillinstreptomycin. ES cells were cultured on mitotically arrested mouse embryonic fibroblast cells in DMEM (Life Technologies), supplemented with 15% v/v FBS (Bodinco BV), 1% v/v penicillin-streptomycin, 1% v/v non-essential amino acids (Life Technologies), 50M 2-mercaptoethanol (ThermoFisher Scientific), and 1,000Uml1 leukemia inhibitor factor (Merck). All cells were cultured at 37C in 5% CO2. Exponentially growing cells were harvested from individual cultures for each sample, and RNA was isolated using the Nucleospin RNA kit (Macherey Nagel). Libraries were generated for each of the three replicates, with exception of one sample where isolation produced RNA with a low RIN score (below 7) and thus was done in duplicate. RNA-sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep kit for Illumina (NEB) combined with the NEBNext rRNA Depletion kit (NEB). Complementary DNA quality and concentrations were assessed using the High Sensitivity dsDNA kit (Agilent) on the Agilent 2100 Bio-Analyzer and the Qubit 2.0 Fluorometer (Life Technologies).