U2OS cells grown in 60 mm plates (50% confluent) were treated with HU and/or ATRi as indicated, and then transfected with 100 ng pNS (the linearized DSBR substrate, Figure 3B ) (30 (link)) using Lipofectamine 2000 and incubated overnight (15 h). Plasmids (5 μl) were isolated using the Qiagen plasmid miniprep kit prior to transformation of XL10-gold ultracompetent Escherichia coli cells (Agilent) following the manufacturer’s protocol. Forty colonies were randomly selected for plasmid sequencing using the CMV-F primer by Genewiz, Inc. Sequences with insertions/deletions of 1–4 nt at the DSB site were scored as NHEJ products, whereas the plasmids with deletion of one of the 5 nt microhomology sequences were scored as products of MMEJ/alt-EJ products, as described (30 (link)). Nonspecific extended deletions (>10 nt) at the 3′ or 5′ end of the DSB were not considered when plotting MMEJ versus NHEJ.
Protocol detail
Quantifying NHEJ and MMEJ in U2OS cells
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Cells
Deletions
Escherichia coli
Gold
Insertions deletions
Lipofectamine 2000
Nhej
Plasmids
Primer
Sequences deletion
Sequences insertions
Corresponding Organization :
Other organizations : Houston Methodist, Texas A&M Health Science Center, The University of Texas MD Anderson Cancer Center, Instituto Conmemorativo Gorgas de Estudios de la Salud, Chinese Academy of Medical Sciences & Peking Union Medical College, Cornell University, New Mexico Cancer Center, University of New Mexico
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Variable analysis
independent variables
- HU (hydroxyurea) treatment
- ATRi (ATR inhibitor) treatment
- Transfection of pNS (the linearized DSBR substrate)
- NHEJ (non-homologous end joining) products
- MMEJ/alt-EJ (microhomology-mediated end joining/alternative end joining) products
- U2OS cells grown in 60 mm plates (50% confluent)
- Incubation time of 15 h after transfection
- Plasmid isolation using Qiagen plasmid miniprep kit
- Transformation of XL10-gold ultracompetent Escherichia coli cells
- Plasmid sequencing using the CMV-F primer by Genewiz, Inc.
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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