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Abstract
Single-molecule DNA fluorescence in situ hybridization (FISH) techniques enable studying the three-dimensional (3D) organization of the genome at the single cell level. However, there is a major unmet need for open access, high quality, curated and reproducible DNA FISH datasets. Here, we describe a dataset obtained by applying our recently developed iFISH method to simultaneously visualize 16 small (size range: 62–73 kilobases, kb) DNA loci evenly spaced on chromosome 2 in human cells, in a single round of hybridization. We show how combinatorial color coding can be used to precisely localize multiple loci in 3D within single cells, and how inter-locus distances scale inversely with chromosome contact frequencies determined by high-throughput chromosome conformation capture (Hi-C). We provide raw images and 3D coordinates for nearly 10,000 FISH dots. Our dataset provides a free resource that can facilitate studies of 3D genome organization in single cells and can be used to develop automatic FISH analysis algorithms.
Highlights
Background & SummaryIn eukaryotic cells, the genome is highly structured and is characterized by distinct chromatin arrangements at different length scales[1,2]
To fill this gap in, here we describe a multi-color DNA fluorescence in situ hybridization (FISH) approach based on our recently developed iFISH method[22], which allows for simultaneous visualization of multiple DNA
We considered in our analysis only G1 cells with two clearly distinct clusters of multi-color DNA FISH (miFISH) dots and excluded signals originating from dual-color probes, when the expected dot pair was further apart than 0.25 or 0.55 μm depending on the dichroic mirror setup
Summary
The genome is highly structured and is characterized by distinct chromatin arrangements at different length scales[1,2]. To fill this gap in, here we describe a multi-color DNA FISH (miFISH) approach based on our recently developed iFISH method[22], which allows for simultaneous visualization of multiple DNA loci in single cells, using combinatorial color indexing (spectral barcoding) of individual loci. We provide two datasets (Datasets and 425,26) containing a single dual-color probe alongside singly labelled probes, used to identify thresholds for calling co-localization events (Fig. 2 and Table 1) Using these data, we identify 740 clusters of signals corresponding to individual alleles of chr[2], which we use to study single chromosome topologies, highlighting extreme cell-to-cell variability in how DNA folds at sub-chromosomal resolution in interphase nuclei (Fig. 3). We provide raw images, 3D FISH dot coordinates after shift and chromatic aberration correction, and all the scripts needed to reproduce the analyses presented here
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