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Protocol publication

[…] For the LacO resection assay, every cell cycle was tracked and quantified individually, including timing of nuclear division, cellular division, Rad52-mCherry focus formation, and LacO/LacI-GFP focus disappearance. Only on-target Rad52 foci (that co-localized with LacO/LacI-GFP for at least 2 frames) were considered, since many DSB events occur throughout the genome spontaneously, especially during S-phase. The number of cells and events used to generate the plots in all Figures is included as . The time between the first frame with an on-target Rad52-mCherry focus and the first frame with complete disappearance of the LacO/LacI-GFP focus is the duration of resection through 3.57 kb (between the HO cut site and the start of the LacO repeats) plus the full 10.3 kb LacO array. All fields from all genotypes were input into custom ImageJ macros that randomized the order of the fields/genotypes, blinded the images by removal of the file names, set the contrast to be identical for every image, and numbered each cell lineage. Each blinded field was then manually assessed for photobleaching of the LacO/LacI-GFP foci in cells without induced DSBs (>80% of all cells) to ensure that disappearance of any LacO/LacI-GFP foci in cells with on-target DSBs was due to resection through the LacO array rather than photobleaching of the GFP signal. Next, using the pre-determined contrast settings for mCherry and GFP channels (to maintain consistency across all images analyzed) individual cells which had on-target DSB events were manually identified, and scored for the first frame of Rad52-mCherry focus appearance and then the first frame in which the LacO/LacI-GFP focus had completely disappeared.For focus intensity plots (e.g. ), quantification was performed in ImageJ on the full 5D image stacks (not maximum intensity projections, which are shown in the image panels throughout for ease of viewing, for example ). Subpixel measurements were made in a cylinder approximating the point spread function surrounding the manually scored subpixel center of the focus. A cylindrical shell surrounding the focus was used for background subtraction in both the GFP and mCherry channels.Raw data were processed, visualized, and analyzed using R, in particular packages dplyr, ggplot2, and broom. Raw data, raw analysis for all individual cells included in plots, complete code, and other supporting materials are publically available on GitHub https://github.com/lelandbr/Leland_King_2018_eLife_Rev7_EndResection (; copy archived at https://github.com/elifesciences-publications/Leland_King_2018_eLife_Rev7_EndResection). […]

Pipeline specifications

Software tools ImageJ, dplyr, Ggplot2
Applications Miscellaneous, Microscopic phenotype analysis
Diseases Deficiency Diseases, Neoplasms
Chemicals Adenosine Diphosphate