Journal: Scientific data

Article Title: Simultaneous visualization of DNA loci in single cells by combinatorial multi-color iFISH.

doi: 10.1038/s41597-022-01139-2

Figure Lengend Snippet: Fig. 1 Overview of miFISH and experimental design used to obtain Dataset 123 and 224. (a) miFISH workflow from probe generation to analysis output. (b) Inter-probe distance between all the 16 miFISH probes along chr2. Letters indicate the fluorescent dye(s) used to label each probe, as indicated on the right. (c) Genomic location of the 16 miFISH probes along chr2 and of the oligos (vertical black bars) composing probe 2.1 as an example. Each probe is displayed as a filled circle either in one (single-color probes) or two (dual-color probes) colors, depending on the combination of fluorescently labeled detection (D) oligos used. In dual-color probes, the oligos are labeled with fluorophores (filled colored circles) in alternating colors, as shown for four consecutive oligos encircled by the dashed square, as an example. Each probe consists of 700 oligos with the structure shown on the top, and the consecutive probes are spaced either 3 Mb or 20 Mb apart as shown below chr2 ideogram and in (c). T, target sequence. F and R, adaptor sequences used to selectively amplify all the oligos composing a probe from a complex oligopool containing 12,000 oligos (see Methods). C, color sequence to which a complementary detection (D) oligo binds. See Supplementary Table 1 for the list of all oligos in each of the 16 probes. (c) Microscopy image showing one example of RPE cell nucleus in which we detected all the 16 miFISH probes displayed in (b) and (c). Scale bar: 10 µm. The smaller panels correspond to the 6 fluorescent dyes shown in (b) imaged separately. The images were deconvolved using the Huygens software, as described in the Methods. Grey, DNA stained with Hoechst 33342.

Article Snippet: We purchased all the oligos as a single 12k oligopool (Twist Biosciences) and produced the 16 probes individually using the iFISH pipeline22.

Techniques: Labeling, Sequencing, Microscopy, Software, Staining