4.7 Article

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

Journal

NATURE PROTOCOLS
Volume 10, Issue 3, Pages -

Publisher

NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.191

Keywords

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Funding

  1. US National Institutes of Health (NIH) Centers of Excellence in Genomic Sciences (CEGS) [P50 HG005550]
  2. National Heart, Blood and Lung Institute (NHBLI) [RC2HL102815]
  3. Allen Institute for Brain Science and by National Institute of Mental Health (NIMH) [MH098977]
  4. NIH [GM080177]
  5. National Science Foundation (NSF) Graduate Research Fellowship [DGE1144152]

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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.

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