Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems

• Published in: Science (American Association for the Advancement of Science) Vol. 371; no. 6528
• Main Authors: Alon, Shahar, Goodwin, Daniel R., Sinha, Anubhav, Wassie, Asmamaw T., Chen, Fei, Daugharthy, Evan R., Bando, Yosuke, Kajita, Atsushi, Xue, Andrew G., Marrett, Karl, Prior, Robert, Cui, Yi, Payne, Andrew C., Yao, Chun-Chen, Suk, Ho-Jun, Wang, Ru, Yu, Chih-Chieh (Jay), Tillberg, Paul, Reginato, Paul, Pak, Nikita, Liu, Songlei, Punthambaker, Sukanya, Iyer, Eswar P. R., Kohman, Richie E., Miller, Jeremy A., Lein, Ed S., Lako, Ana, Cullen, Nicole, Rodig, Scott, Helvie, Karla, Abravanel, Daniel L., Wagle, Nikhil, Johnson, Bruce E., Klughammer, Johanna, Slyper, Michal, Waldman, Julia, Jané-Valbuena, Judit, Rozenblatt-Rosen, Orit, Regev, Aviv, Church, George M., Marblestone, Adam H., Boyden, Edward S., Ali, H. R., Al Sa’d, M., Alon, S., Aparicio, S., Battistoni, G., Balasubramanian, S., Becker, R., Bodenmiller, B., Boyden, E. S., Bressan, D., Bruna, A., Burger, Marcel, Caldas, C., Callari, M., Cannell, I. G., Casbolt, H., Chornay, N., Cui, Y., Dariush, A., Dinh, K., Emenari, A., Eyal-Lubling, Y., Fan, J., Fatemi, A., Fisher, E., González-Solares, E. A., González-Fernández, C., Goodwin, D., Greenwood, W., Grimaldi, F., Hannon, G. J., Harris, O., Harris, S., Jauset, C., Joyce, J. A., Karagiannis, E. D., Kovačević, T., Kuett, L., Kunes, R., Küpcü Yoldaş, A., Lai, D., Laks, E., Lee, H., Lee, M., Lerda, G., Li, Y., McPherson, A., Millar, N., Mulvey, C. M., Nugent, F., O'Flanagan, C. H., Paez-Ribes, M., Pearsall, I., Qosaj, F., Roth, A. J., Rueda, O. M., Ruiz, T., Sawicka, K.
• Format: Journal Article
• Language: English
• Published: United States The American Association for the Advancement of Science 29.01.2021
• Subjects: Alternative splicing; Animals; Biopsy; Breast cancer; Breast Neoplasms - immunology; Breast Neoplasms - pathology; Cancer; Chemical reactions; Compartments; Context; Dendrites; Dendritic Spines; Dendritic transport; Female; Fluorescence; Gene expression; Gene Expression Profiling - methods; Gene mapping; Gene sequencing; Genes; Hippocampus; Humans; Hydrogels; Immune system; Interrogation; Introns; Localization; Mapping; Medical imaging; Metastases; Metastasis; Mice; Microscopes; Microscopy; Molecular Imaging - methods; Multiplexing; Neuroimaging; Neurons; Polymers; Pyramidal cells; Ribonucleic acid; RNA; Sequence Analysis, RNA - methods; Single-Cell Analysis - methods; Spatial discrimination; Spatial resolution; Tissues; Transcription factors; Transcripts (Written Records); Tumor cells; Tumors; Visual Cortex; Visual observation; Visualization
• ISSN: 0036-8075; 1095-9203; 1095-9203
• DOI: 10.1126/science.aax2656

Identifying where specific RNAs occur within a cell or tissue has been limited by technology and imaging capabilities. Expansion microscopy has allowed for better visualization of small structures by expanding the tissues with a polymer- and hydrogel-based system. Alon et al. combined expansion microscopy with long-read in situ RNA sequencing, resulting in a more precise visualization of the location of specific transcripts. This method, termed “ExSeq” for expansion sequencing, was used to detect RNAs, both new transcripts and those previously demonstrated to localize to neuronal dendrites. Unlike other in situ sequencing methods, ExSeq does not target sets of genes. This technology thus unites spatial resolution, multiplexing, and an unbiased approach to reveal insights into RNA localization and its physiological roles in developing and active tissue. Science , this issue p. eaax2656 An adaptation of expansion microscopy allows in situ sequencing and multiplexed subcellular mapping of RNAs. Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.