Fluorescent in situ sequencing

Fluorescent in situ sequencing (FISSEQ) is a method of sequencing a cell's RNA while it remains in tissue or culture using next-generation sequencing. Fluorescent in situ sequencing (FISSEQ) is a method of sequencing a cell's RNA while it remains in tissue or culture using next-generation sequencing. FISSEQ combines the spatial context of RNA-FISH and the global transcriptome profiling of RNA-seq. FISSEQ preserves the tissue allowing single molecule in situ RNA localization. The foundation of the method is a novel nucleic acid sequencing library construction method that stably cross-links cDNA amplicons within biological samples. Sequencing data is then generated through an intensive interleaved microscopy and biochemistry protocol and subsequent image processing and bioinformatics. FISSEQ is compatible with diverse sample types including cell culture, tissue sections, and whole mount embryos. FISSEQ is an example of an extremely dense form of in-situ nucleic acid readout: every letter along the RNA chain is read. Thus, barcodes for FISSEQ can be packed into a short string of DNA, as short as 15-20 nucleotides long for the mouse brain or 5 nucleotides for targeted cancer gene panels.