DNA Fiber Assay for Replication Dynamics

DNA fiber assay was carried out as described previously (Castillo et al. 2014 (link)). Briefly, cells were pulse-labeled with 100 µM CldU and 250 µM IdU for 30 min. After labeling and treatment, cells were harvested, lysed, and spread on microscope slides. Immunofluorescence was carried out using α-IdU/α-BrdU (BD Biosciences) and α-CldU/α-BrdU (Abcam) and secondary antibodies, including Alexa fluor 488 (green) and Alexa fluor 555 (Invitrogen). Images were taken using a Nikon 80 microscope with a 60× lens and analyzed using ImageJ software. Statistics were calculated using Prism software.

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Xu S., Wu X., Wu L., Castillo A., Liu J., Atkinson E., Paul A., Su D., Schlacher K., Komatsu Y., You M.J, & Wang B. (2017). Abro1 maintains genome stability and limits replication stress by protecting replication fork stability. Genes & Development, 31(14), 1469-1482.

Publication 2017

α-IdU/α-BrdU α-CldU/α-BrdU Alexa fluor 488 Alexa fluor 555

Alexa fluor 488 Alexa fluor 555 Antibodies Assay Brdu Cells Fiber Immunofluorescence Lens Microscope Prism Pulse

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Pulse-labeling with 100 µM CldU and 250 µM IdU for 30 min

dependent variables

Immunofluorescence using α-IdU/α-BrdU and α-CldU/α-BrdU antibodies
Microscopy and image analysis using ImageJ software

control variables

Cell harvesting, lysis, and spreading on microscope slides
Secondary antibodies, including Alexa fluor 488 (green) and Alexa fluor 555

controls

No positive or negative controls were explicitly mentioned in the provided information.

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