# Allele-level visualization of transcription and chromatin by high-throughput imaging

## TL;DR

This paper introduces high-throughput imaging methods to visualize DNA and RNA at the allele level, enabling detailed study of chromatin and gene expression in single cells.

## Contribution

The study presents optimized DNA/RNA FISH protocols in a high-throughput format for allele-level visualization of chromatin and transcription.

## Key findings

- Optimized DNA/RNA FISH protocols enable accurate detection of chromatin loci and gene expression status in large cell populations.
- The methods successfully visualized MYC and EGFR DNA and RNA in multiple cell types with allele-level resolution.
- The radial positions of active and inactive MYC and EGFR alleles were determined using these approaches.

## Abstract

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.