Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2015-03

AUTHORS

Je Hyuk Lee, Evan R Daugharthy, Jonathan Scheiman, Reza Kalhor, Thomas C Ferrante, Richard Terry, Brian M Turczyk, Joyce L Yang, Ho Suk Lee, John Aach, Kun Zhang, George M Church

ABSTRACT

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. More... »

PAGES

442-458

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nprot.2014.191

    DOI

    http://dx.doi.org/10.1038/nprot.2014.191

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1019268408

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/25675209


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