A high-throughput DNA FISH protocol to visualize genome regions in human cells

Summary

Here, we describe an end-to-end high-throughput imaging protocol to visualize genomic loci in cells at high throughput using DNA fluorescence in situ hybridization, automated microscopy, and computational analysis. This is particularly useful for quantifying patterns of heterogeneity in relative gene positioning or differences within subpopulations of cells. We focus on important experimental design and execution steps in this one-week protocol, suggest ways to ensure and verify data quality, and provide practical solutions to common problems.

For complete details on the generation and use of this protocol, please refer to Finn et al. (2019).

Graphical abstract

Highlights

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  DNA fluorescence in situ hybridization technique for high-content imaging

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  Optimized for use in a 384-well plate format with cultured cells

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  One-week protocol to assess thousands of cells per condition in tens of conditions

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  Thorough discussion of troubleshooting for new probes, probe types, and cell types

Abstract

Here, we describe an end-to-end high-throughput imaging protocol to visualize genomic loci in cells at high throughput using DNA fluorescence in situ hybridization, automated microscopy, and computational analysis. This is particularly useful for quantifying patterns of heterogeneity in relative gene positioning or differences within subpopulations of cells. We focus on important experimental design and execution steps of this one-week protocol, suggest ways to ensure and verify data quality, and provide practical solutions to common problems.