Spontaneous mitotic recombination assay was described before [21 (link)]. Briefly, cells carrying HR-Flex or HR-Luc reporter were pre-sorted by FACS (fluorescence-activated cell sorting) to clear background and cultured for indicated days. The mitotic recombination frequency (EGFP-positive events) then was determined by FACS analysis using a BD Accuri C6 flow cytometer and accompanying data analysis software (CFlow, Becton-Dickinson).
Plasmid stability assay was performed as described [21 (link)]. Briefly, Flex1 or Luc containing pCEP4 plasmids carrying Epstein-Barr virus (EBV) replication origins and nuclear antigen (encoded by the EBNA-1 gene) to permit extrachromosomal replication in human cells and a hygromycin marker [21 (link)], were transfected into the cells expressing wide-type or BLM mutants by lipofectamine 2000 (Thermo Fisher Scientific), followed by hygromycin selection. The endogenous BLM was knocked down by shRNA, and cells were cultured in the absence of hygromycin for 10 days. Then hygromycin was re-introduced into the medium to determine the percentage of cells retaining the plasmids. Sequencing of Flex1 on pCEP4-Flex1 was performed after propagating pCEP4-Flex1 10 days in U2OS cells with or without expressing BLM-shRNAs.
Plasmid stability assay was performed as described [21 (link)]. Briefly, Flex1 or Luc containing pCEP4 plasmids carrying Epstein-Barr virus (EBV) replication origins and nuclear antigen (encoded by the EBNA-1 gene) to permit extrachromosomal replication in human cells and a hygromycin marker [21 (link)], were transfected into the cells expressing wide-type or BLM mutants by lipofectamine 2000 (Thermo Fisher Scientific), followed by hygromycin selection. The endogenous BLM was knocked down by shRNA, and cells were cultured in the absence of hygromycin for 10 days. Then hygromycin was re-introduced into the medium to determine the percentage of cells retaining the plasmids. Sequencing of Flex1 on pCEP4-Flex1 was performed after propagating pCEP4-Flex1 10 days in U2OS cells with or without expressing BLM-shRNAs.
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