Visualizing RNA-DNA Hybrids in Cells

Chromosome spread from cells grown to mid-log phase in YPAD were prepared and labeled with the monoclonal antibody S9.6 and immunodetected with Cy3- conjugated goat anti-mouse antibody (Jackson Laboratories, #115-165-003 as described [79 (link)]). Slides were mounted with 50 μl of VectaShield (Vector Laboratories, CA) with 1x DAPI and sealed with nail polish. For each replicate (n>3), between 150 and 250 nuclei were visualized and manually counted to obtain the fraction with detectable RNA-DNA hybrids.

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Lafuente-Barquero J., Luke-Glaser S., Graf M., Silva S., Gómez-González B., Lockhart A., Lisby M., Aguilera A, & Luke B. (2017). The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage. PLoS Genetics, 13(12), e1007136.

Publication 2017

Cy3- conjugated goat anti-mouse antibody VectaShield

Anti antibody Cells Chromosome Dapi Goat Hybrids Monoclonal antibody Mouse Nail Nuclei Replicate Vector

Corresponding Organization : University of Copenhagen

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Variable analysis

independent variables

Chromosome spread from cells grown to mid-log phase in YPAD

dependent variables

Fraction of nuclei with detectable RNA-DNA hybrids

control variables

Cells grown to mid-log phase in YPAD
Monoclonal antibody S9.6 used for labeling
Cy3-conjugated goat anti-mouse antibody used for immunodetection
VectaShield with 1x DAPI used for mounting and sealing slides

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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