Fluorescent In Situ Sequencing

**Semantic type:** Laboratory Procedure

**Definition:** A fluorescent next generation sequencing technique that can analyze the transcriptome in fixed cell or tissue samples in situ, in a way that preserves the samples architecture and allows for spatial localization of mRNA expression in the sample. The sample is fixed on a glass slide and mRNA are converted to cDNA by in situ reverse transcription using either random or targeted primers. Residual RNA is removed and the cDNA fragments are cross-linked to the original spot by bis-succinimide ester-activated PEG using primary amine linkers. Each cDNA is amplified using rolling-circle amplification yielding a single molecule bearing multiple copies of the original sequence. The amplicons are crosslinked and subjected to partition sequencing using fluorescent probes. Sequences can be read using high resolution confocal microscopy.

**Synonyms:** - FISSEQ