Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues
Open accessAbstract
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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Emerging spatial transcriptomics platforms allow high-resolution, multiplexed mapping of RNA molecules within intact 3D cultures.
“Emerging spatial transcriptomics platforms, such as MERSCOPE (Vizgen), FISSEQ, ExSeq, Slide-seq, and 10x Genomics Visium, now allow for high-resolution, multiplexed mapping of RNA molecules within intact 3D cultures [199,200,201].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [201]
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- Authors
- Lee, J. H., Daugharthy, E. R., Scheiman, J., Kalhor, R., Ferrante, T. C., Terry, R., Turczyk, B. M., Yang, J. L., Lee, H. S., Aach, J., Zhang, K., & Church, G. M.
- Affiliations
- Harvard University (US)
- University of California San Diego (US)
- Corresponding author
- Lee, Je Hyuk
- Church, George M
- Venue
- Nature Protocols
- Published
- 2015-02-12
- Volume
- 10
- Issue
- 3
- Pages
- 442-458
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC4327781
- OpenAlex
- W2112138633
- Cited by
- 529
- References
- 10
- Citations by year
- 2026: 21 · 2025: 46 · 2024: 69 · 2023: 54 · 2022: 57 · 2021: 54 · 2020: 54 · 2019: 38 · 2018: 56 · 2017: 38 · 2016: 27 · 2015: 13 · 2014: 1
- FWCI
- 19.40
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Heart, Lung, and Blood Institute (RC2HL102815)
- National Institute of Mental Health (MH098977, U01MH098977)
- National Human Genome Research Institute (P50HG005550)
- National Institute of General Medical Sciences (T32GM080177, GM080177)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- spatial transcriptomics, mRNA localization, library construction, tissue architecture preservation
- MeSH terms
- Fluorescence, RNA, Messenger, Sequence Analysis, RNA, DNA, Complementary, Microscopy, Confocal, Gene Expression Profiling
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