The paper introduces a method called Fluorescent In Situ RNA Sequencing (FISSEQ), which allows for the localization of RNA transcripts within cells in situ. FISSEQ involves stably cross-linking cDNA amplicons within a biological sample and sequencing them using next-generation sequencing technologies. The method was tested on human primary fibroblasts using a simulated wound healing assay, and it was found to be compatible with tissue sections and whole-mount embryos. FISSEQ overcomes the limitations of optical resolution and noisy signals in single-molecule detection, enabling the detection of genetic elements such as gene transcripts and molecular barcodes. The platform can be used to investigate cellular phenotype, gene regulation, and environmental factors in situ. The authors also developed partition sequencing to control the signal density and improve the accuracy of RNA localization. FISSEQ correlates well with RNA-seq, except for genes involved in RNA and protein processing, and it can be used to identify cell types based on gene expression profiles. The method shows promise for advancing the understanding of gene expression and its spatial organization in various biological contexts.The paper introduces a method called Fluorescent In Situ RNA Sequencing (FISSEQ), which allows for the localization of RNA transcripts within cells in situ. FISSEQ involves stably cross-linking cDNA amplicons within a biological sample and sequencing them using next-generation sequencing technologies. The method was tested on human primary fibroblasts using a simulated wound healing assay, and it was found to be compatible with tissue sections and whole-mount embryos. FISSEQ overcomes the limitations of optical resolution and noisy signals in single-molecule detection, enabling the detection of genetic elements such as gene transcripts and molecular barcodes. The platform can be used to investigate cellular phenotype, gene regulation, and environmental factors in situ. The authors also developed partition sequencing to control the signal density and improve the accuracy of RNA localization. FISSEQ correlates well with RNA-seq, except for genes involved in RNA and protein processing, and it can be used to identify cell types based on gene expression profiles. The method shows promise for advancing the understanding of gene expression and its spatial organization in various biological contexts.