iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture

Abstract

DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.

Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells

“…Single-molecule RNA fluorescence in situ hybridization. Custom mouse specific probes for single-molecule fluorescence in situ hybridization of gene pairs of interest were designed and produced in-house using an analytic software and production pipeline 61 . Each probe consists of 42-86 oligos (47,51,63,42, and 86 oligos for the probe targeting the Adam19, Adam23, Dnmt3, Mme, and Tmem2 respectively) and each oligo consists of four parts (from 5′ to 3′): (1) a 20 nt adapter, C, for probe visualization; (2) a 20 nt adapter, F, for PCR amplification during probe synthesis; (3) a 30 nt T sequence complementary to the target; and (4) a 20 nt adapter, R, for PCR amplification during probe synthesis.…”

“…The T sequences for all the probes as well as the transcript variant which they are targeting can be found in Supplementary Data 3. The C, F, and R adapter sequences were designed as explained in Gelali et al 61 with the difference that the sequences were checked for orthogonally against the mouse genome. The probes were produced using a pipeline for large-scale enzymatic production of hundreds of probes in parallel that is describe in details in (Gelali et al 61 ).…”

“…The C, F, and R adapter sequences were designed as explained in Gelali et al 61 with the difference that the sequences were checked for orthogonally against the mouse genome. The probes were produced using a pipeline for large-scale enzymatic production of hundreds of probes in parallel that is describe in details in (Gelali et al 61 ). More specifically, the F and R sequences are used as barcodes for the selective amplification of the desired oligonucleotides from an array-synthesized complex oligo pool (containing thousands of different species of oligonucleotides).…”

Radial Organization in the Mammalian Nucleus

“…One limitation of these studies is that only a few chromosomes were investigated in cultured cells of a single cell type (mouse embryonic fibroblasts in (Luperchio et al, 2018) and human fibroblasts in ). In the future, application of high-throughput FISH techniques, together with novel ways for visualizing the internal structure of CTs, such as 'chromosome spotting' (Gelali et al, 2019), will enable us to draw a refined portrait of the internal radial organization of all chromosomes, in many different cell types.…”

Evidence for ABL Amplification in Multiple Myeloma and Its Role in Treatment

“…DNA FISH techniques have become increasingly popular among genome biologists, now widely accepted as the primary methodology for the validation of Hi-C results. Thanks to the direct observation of the three-dimentional (3D) genome architecture in a manner that is complementary to chromosome conformation capture methods such as Hi-C [11,12], as well as FISH probes produced, FISH is a versatile and expandable resource to study genome architecture, which can greatly facilitate the research and diagnostics of diseases.…”