Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections


Spatial resolution of gene expression enables gene expression events to be pinpointed to a specific location in biological tissue. Spatially resolved gene expression in tissue sections is traditionally analyzed using immunohistochemistry (IHC) or in situ hybridization (ISH). These technologies are invaluable tools for pathologists and molecular biologists; however, their throughput is limited to the analysis of only a few genes at a time. Recent advances in RNA sequencing (RNA-seq) have made it possible to obtain unbiased high-throughput gene expression data in bulk. Spatial Transcriptomics combines the benefits of traditional spatially resolved technologies with the massive throughput of RNA-seq. Here, we present a protocol describing how to apply the Spatial Transcriptomics technology to mammalian tissue. This protocol combines histological staining and spatially resolved RNA-seq data from intact tissue sections. Once suitable tissue-specific conditions have been established, library construction and sequencing can be completed in ~5–6 d. Data processing takes a few hours, with the exact timing dependent on the sequencing depth. Our method requires no special instruments and can be performed in any laboratory with access to a cryostat, microscope and next-generation sequencing.

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Fig. 1: Overview of the protocol.
Fig. 2: Bright-field and fluorescence images demonstrating the tissue-optimization stage on mouse olfactory bulb.
Fig. 3: Typical results seen during the library construction steps.
Fig. 4: Flowchart of the experimental part of the Spatial Transcriptomics approach.
Fig. 5: Overview of the computational steps for Spatial Transcriptomics.
Fig. 6: Bright-field images of expected tissue morphology.
Fig. 7: Number and spatial distribution of detected genes and unique transcripts from typical experiments.


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We thank M. Asp for valuable contributions. This work was supported by the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Swedish Foundation for Strategic Research, the Swedish Cancer Society, the Karolinska Institutet, Ragnar Söderbergs Stiftelse, Torsten Söderbergs Stiftelse, Tobias Stiftelsen, the Åke Wiberg Foundation, StratRegen and the Jeansson Foundations. P.L.S. was supported by a grant from the Swedish Research Council. We also thank the Swedish National Genomics Infrastructure at SciLifeLab for providing sequencing assistance and infrastructure, and the Swedish National Infrastructure for Computing–Uppsala Multidisciplinary Center for Advanced Computational Science and Bioinformatics Long-Term Support for providing computational assistance.

Author information

F.S. and P.L.S. developed the method and wrote the manuscript; A.M. and S.V. contributed to the protocol development; J.F.N. developed computational methods for the method; F.S. and A.M. wrote the protocol; and J.L., P.L.S. and J.F. planned the study.

Correspondence to Patrik L. Ståhl or Joakim Lundeberg.

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Competing interests

J.F., J.L., P.L.S. and F.S. are authors on patents owned by Spatial Transcriptomics AB covering the technology. The remaining authors declare no competing interests.

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Key references using this protocol

Ståhl, P.L. et al. Science 353, 78–82 (2016):

Jemt, A. et al. Sci. Rep. 7, 41109 (2017):

Asp, M. et al. Sci. Rep. 7, 12941 (2017):

Berglund, E. et al. Nat. Commun. 9, 2419 (2018):

An extension to this protocol adapted for use on plant material

Giacomello, S. & Lundeberg, J. Nat. Protoc. (2018):

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Salmén, F., Ståhl, P.L., Mollbrink, A. et al. Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections. Nat Protoc 13, 2501–2534 (2018).

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