The in situ sequencing (ISS) facility unit provides spatially resolved gene expression data for panels of genes at subcellular resolution. The technique has been developed in the lab of Mats Nilsson who has pioneered the field of generating in situ gene expression and mutation profiles (Ke, R., et al. 2013 Nature Methods 10, 857-860).
Unique Features of ISS
In situ sequencing enables localisation and quantification of more than 100 transcripts simultaneously with subcellular resolution in a single tissue section in a single experiment.
Tissue/cell line samples on standard microscopy slides are pre-treated to generate cDNA in situ. A set of genes are targeted with a custom designed padlock probe library. Probes that have specifically interacted with the targeted transcripts are amplified by Rolling Circle Amplification (RCA) reaction, and the specific amplification generates signals with high signal to noise ratio.
Specific barcode sequence in the probe for each targeted gene is decoded by sequential hybridization reaction and imaging cycles. The signals are detected by fluorescently labelled oligonucleotide libraries. ISS can be used to detect up to a few hundred genes per sample. Wide-field imaging enables high throughput.
After the image acquisition, images are processed with the analysis pipeline. CSV file is generated for x-y coordinates map of each signal, and each cell nucleus.
The method has been successfully demonstrated for a variety of applications such as mapping of
across different type of sections including fresh frozen, PFA-fixed and FFPE in various tissues.
In Situ Sequencing
171 21 Solna
In Situ Sequencing
171 65 Solna
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