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

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

Research output: Contribution to journalArticlepeer-review

202 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)442-458
Number of pages17
JournalNature protocols
Volume10
Issue number3
DOIs
StatePublished - Mar 2 2015
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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