The image analysis protocol for DNA/RNA/IF-seqFISH has been published in Methods Mol. Biol. (Computational Methods for 3D Genome Analysis, edited by Ryuichi Nakato)!
https://link.springer.com/protocol/10.1007/978-1-0716-4136-1_24#DOI
Image Analysis Protocol for DNA/RNA/Immunofluorescence (IF)-seqFISH Data
Hiroaki Ohishi & Hiroshi Ochiai
Abstract
Imaging-based spatial multi-omics technologies enable the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level. This allows for detailed observation of biological phenomena, including transcription machinery within cells and tissues. This chapter provides an in-depth explanation of the principles behind these technologies, focusing on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). The chapter also offers a comprehensive, step-by-step image analysis protocol, covering aspects such as image preprocessing, spot detection, and data visualization. For practical use, complete Jupyter Notebook codes are available on GitHub (https://github.com/Ochiai-Lab/seqFISH_analysis).