A new method called FISSEQ (Fluorescent In Situ Sequencing) enables highly multiplexed, subcellular RNA sequencing in situ. This technique allows for the localization of RNA transcripts within cells, tissues, and organisms with high spatial resolution. FISSEQ uses fluorescent in situ sequencing to generate cDNA amplicons, which are then sequenced to identify gene expression and localization. The method is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. The process involves reverse transcription of RNA in fixed cells with tagged random hexamers, followed by circularization and rolling circle amplification (RCA) of the cDNA. The amplicons are then cross-linked and sequenced using a method called SOLiD sequencing by ligation. This allows for the identification of RNA transcripts and molecular barcodes, enabling the investigation of cellular phenotype, gene regulation, and environment in situ. FISSEQ was tested on human primary fibroblasts and showed high accuracy in identifying gene expression and localization. The method was also used to study the effects of simulated wound healing on gene expression, revealing differences in gene expression between fibroblasts in different media. The results showed that FISSEQ can detect a wide range of genes, including those involved in extracellular matrix, bone development, and skin development. FISSEQ was compared to RNA-seq and gene expression arrays, showing good correlation for moderately expressed genes, but lower correlation for genes with low or high expression levels. The method also showed that nuclear RNA is more likely to be non-coding, and antisense mRNA is more likely to be nuclear. FISSEQ was also used to study splicing junctions of the fibronectin gene, revealing differences in splicing patterns between different cell types. Overall, FISSEQ provides a powerful tool for transcriptome-wide RNA sequencing in situ, enabling the study of gene expression and localization at a high spatial resolution. The method has the potential to be used for identifying cell types based on gene expression profiles in situ, and may be combined with in situ mutation detection in a high-throughput manner. FISSEQ is a promising technique for advancing our understanding of gene expression and regulation in biological systems.A new method called FISSEQ (Fluorescent In Situ Sequencing) enables highly multiplexed, subcellular RNA sequencing in situ. This technique allows for the localization of RNA transcripts within cells, tissues, and organisms with high spatial resolution. FISSEQ uses fluorescent in situ sequencing to generate cDNA amplicons, which are then sequenced to identify gene expression and localization. The method is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. The process involves reverse transcription of RNA in fixed cells with tagged random hexamers, followed by circularization and rolling circle amplification (RCA) of the cDNA. The amplicons are then cross-linked and sequenced using a method called SOLiD sequencing by ligation. This allows for the identification of RNA transcripts and molecular barcodes, enabling the investigation of cellular phenotype, gene regulation, and environment in situ. FISSEQ was tested on human primary fibroblasts and showed high accuracy in identifying gene expression and localization. The method was also used to study the effects of simulated wound healing on gene expression, revealing differences in gene expression between fibroblasts in different media. The results showed that FISSEQ can detect a wide range of genes, including those involved in extracellular matrix, bone development, and skin development. FISSEQ was compared to RNA-seq and gene expression arrays, showing good correlation for moderately expressed genes, but lower correlation for genes with low or high expression levels. The method also showed that nuclear RNA is more likely to be non-coding, and antisense mRNA is more likely to be nuclear. FISSEQ was also used to study splicing junctions of the fibronectin gene, revealing differences in splicing patterns between different cell types. Overall, FISSEQ provides a powerful tool for transcriptome-wide RNA sequencing in situ, enabling the study of gene expression and localization at a high spatial resolution. The method has the potential to be used for identifying cell types based on gene expression profiles in situ, and may be combined with in situ mutation detection in a high-throughput manner. FISSEQ is a promising technique for advancing our understanding of gene expression and regulation in biological systems.