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.

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Acknowledgements

This study was funded by US National Institutes of Health (NIH) Centers of Excellence in Genomic Sciences (CEGS) grant no. P50 HG005550. J.H.L. and co-workers were funded by National Heart, Blood and Lung Institute (NHBLI) grant no. RC2HL102815, by the Allen Institute for Brain Science and by National Institute of Mental Health (NIMH) grant no. MH098977. E.R.D. was funded by NIH grant no. GM080177 and by National Science Foundation (NSF) Graduate Research Fellowship grant no. DGE1144152.

Author information

Author notes

    • Je Hyuk Lee
    •  & Evan R Daugharthy

    These authors contributed equally to this work.

Affiliations

  1. Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Je Hyuk Lee
    • , Evan R Daugharthy
    • , Jonathan Scheiman
    • , Thomas C Ferrante
    • , Richard Terry
    • , Brian M Turczyk
    •  & George M Church
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Evan R Daugharthy
    • , Jonathan Scheiman
    • , Reza Kalhor
    • , Joyce L Yang
    • , John Aach
    •  & George M Church
  3. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Evan R Daugharthy
  4. Department of Electrical and Computer Engineering, University of California San Diego, California, USA.

    • Ho Suk Lee
  5. Department of Bioengineering, University of California San Diego, La Jolla, California, USA.

    • Kun Zhang

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Contributions

J.H.L. and E.R.D. conceived FISSEQ library construction, sequencing, image analysis and bioinformatics. J.S., R.K., J.L.Y., B.M.T., H.S.L. and J.A. provided key feedbacks during the FISSEQ method development. R.T. and T.C.F. assisted with automated microscopy and image analysis. K.Z. and G.M.C. oversaw the project. J.H.L. wrote the paper, and E.R.D. wrote the FISSEQ software.

Competing interests

Potential conflicts of interest for G.M.C. are listed on http://arep.med.harvard.edu/gmc/tech.html. Other authors have no conflicts of interest.

Corresponding authors

Correspondence to Je Hyuk Lee or George M Church.

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DOI

https://doi.org/10.1038/nprot.2014.191

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