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Nat Protoc. 2015 Mar;10(3):442-58. doi: 10.1038/nprot.2014.191. Epub 2015 Feb 12.

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

Author information

1
Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA.
2
1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
3
1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
4
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
5
Department of Electrical and Computer Engineering, University of California San Diego, California, USA.
6
Department of Bioengineering, University of California San Diego, La Jolla, California, USA.

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.

PMID:
25675209
PMCID:
PMC4327781
DOI:
10.1038/nprot.2014.191
[Indexed for MEDLINE]
Free PMC Article

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