Visualizing epigenetic modifications and their spatial proximities in single cells using three DNA-encoded amplifying FISH imaging strategies: BEA-FISH, PPDA-FISH and Cell-TALKING.

Epigenetic modifications and spatial proximities of nucleic acids and proteins play important roles in regulating physiological processes and disease progression. Currently available cell imaging methods, such as fluorescence in situ hybridization (FISH) and immunofluorescence, struggle to detect low-abundance modifications and their spatial proximities. Here we describe a step-by-step protocol for three DNA-encoded amplifying FISH-based imaging strategies to overcome these challenges for varying applications: base-encoded amplifying FISH (BEA-FISH), pairwise proximity-differentiated amplifying FISH (PPDA-FISH) and cellular macromolecules-tethered DNA walking indexing (Cell-TALKING). They all use the similar core principle of DNA-encoded amplification, which transforms different nonsequence molecular features into unique DNA barcodes for in situ rolling circle amplification and FISH analysis. This involves three key reactions in fixed cell samples: target labeling, DNA encoding and rolling circle amplification imaging. Using this protocol, these three imaging strategies achieve in situ counting of low-abundance modifications alone, the pairwise proximity-differentiated visualization of two modifications and the exploration of multiple modifications around one protein (one-to-many proximity), respectively. Low-abundance modifications, including 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil and 5-formyluracil, are clearly visualized in single cells. Various combinatorial patterns of nucleic acid modifications and/or histone modifications are found. The whole protocol takes ~2-4 d to complete, depending on different imaging applications.