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RESEARCH ◥ proteins and lipids. This form of hydrogel- RESEARCH ARTICLE SUMMARY tissue chemistry replots amplicons onto an op- tically transparent hydrogel coordinate system; then, to identify and quantify RNA species- BIOTECHNOLOGY abundance manifested by DNA amplicons, the identity of each species is encoded as a five-base barcode and read out by means of an in situ Three-dimensional intact-tissue sequencing method that decodes DNA sequence in multicolor fluorescence. Using a new two-base sequencing of single-cell sequencing scheme (SEDAL), STARmap was found to simultaneously detect more than 1000 transcriptional states genes over six imaging cycles, in which sequencing errors in any cycle cause misdecoding and Xiao Wang*, William E. Allen*, Matthew A. Wright, Emily L. Sylwestrak, are effectively rejected. Nikolay Samusik, Sam Vesuna, Kathryn Evans, Cindy Liu, Charu Ramakrishnan, Jia Liu, Garry P. Nolan†, Felice-Alessio Bava†, Karl Deisseroth† RESULTS: We began by (i) detecting and quan- tifying a focused 160-gene set (including cell type markers and activity-regulated genes) simul- INTRODUCTION: Single-cell RNA sequencing molecule). Other pioneering in situ sequencing taneously in mouse primary visual cortex; (ii) has demonstrated that both stable cell types methods (via padlock probes and fluorescent ◥ clustering resulting per-cell Downloaded from and transient cell states can be discovered and in situ sequencing) use enzymatic amplification, ON OUR WEBSITE gene expression patterns defined by transcriptomes. In situ transcrip- thus achieving high intensity but with room Read the full article into a dozen distinct inhib- tomic methods can map both RNA quantity to improve on efficiency. at http://dx.doi. itory, excitatory, and non- and position; however, it remains challenging org/10.1126/ neuronal cell types; and to simultaneously satisfy key technological re- RATIONALE: We have developed, validated, science.aat5691 (iii) mapping the spatial .................................................. quirements such as efficiency, signal intensity, and applied STARmap (spatially-resolved tran- distribution of all of these http://science.sciencemag.org/ accuracy, scalability to large gene numbers, and script amplicon readout mapping). STARmap cell types across layers of cortex. For validation, applicability to three-dimensional (3D) volumes. begins with labeling of cellular RNAs by pairs per-cell-type gene expression was found to cor- Well-established single-molecule fluorescence of DNA probes followed by enzymatic amplifica- relate well both with in situ hybridization re- in situ hybridization (FISH) approaches (such tion so as to produce a DNA nanoball (amplicon), sults and with single-cell RNA sequencing, and as MERFISH and seqFISH) have high detection which eliminates background caused by mis- widespread up-regulation of activity-regulated efficiency but require long RNA species (more labeling of single probes. Tissue can then be genes was observed in response to visual stim- than 1000 nucelotides) and yield lower intensity transformed into a 3D hydrogel DNA chip by ulation. We next applied STARmap to a higher than that of enzymatic amplification methods anchoring DNA amplicons via an in situ– cognitive area (the medial prefrontal cortex) and (tens versus thousands of fluorophores per RNA synthesized polymer network and removing discovered a more complex distribution of cell types. Last, we extended STARmap to much larger on January 3, 2020 Intact tissue Targeted in situ RNA sequencing approach numbers of genes and spatial scales; we measured 1020 genes simultaneously in sections— Cell SNAIL probes Hydrogel-tissue chemistry DNA obtaining results concordant with the 160-gene Barcodes DNA-gel amplicons set—and measured 28 genes across millimeter- mRNA Amplify scale volumes encompassing ~30,000 cells, re- vealing 3D patterning principles that jointly mRNA 3D sequencing characterize a broad and diverse spectrum of in the gel SEDAL sequencing with error-reduction >1000 genes mapped over cell types. six cycles 5-nt code error-check CONCLUSION: STARmap combines hydrogel- ing tissue chemistry and in situ DNA sequencing Decode to achieve intact-tissue single-cell measurement of expression of more than a thousand genes. STARmap: discovery and distribution of cell types in 3D In the future, combining this intact-system gene Cell types expression measurement with complementary cellular-resolution methodologies (with which STARmap is designed to be compatible)— Genes including in vivo activity recording, optogenetic causal tests, and anatomical connectivity in the Cells same cells—will help bridge molecular, cellular, Single-cell analyses of gene and circuit scales of neuroscience.▪ expression STARmap for 3D transcriptome imaging and molecular cell typing. STARmap is an in situ The list of author affiliations is available in the full article online. RNA-sequencing technology that transforms intact tissue into a 3D hydrogel-tissue hybrid and *These authors contributed equally to this work. measures spatially resolved single-cell transcriptomes in situ. Error- and background-reduction †Corresponding author. Email: [email protected] (K.D.); [email protected] (F.-A.B.); [email protected] (G.P.N.) mechanisms are implemented at multiple layers, enabling precise RNA quantification, spatially Cite this article as X. Wang et al., Science 361, eaat5691 (2018). resolved cell typing, scalability to large gene numbers, and 3D mapping of tissue architecture. DOI: 10.1126/science.aat5691 Wang et al., Science 361, 380 (2018) 27 July 2018 1of1 RESEARCH ◥ possible to perform 3D in situ sequencing with- RESEARCH ARTICLE in such a tissue-hydrogel formulation—leveraging the crucial attendant properties of optical trans- parency, reduced background, elevated diffusion BIOTECHNOLOGY rate, and greater mechanical stability. We achieved this goal with the development and application of a sequencing-based method (spatially-resolved Three-dimensional intact-tissue transcript amplicon readout mapping, or STARmap) for targeted 3D in situ transcriptomics in intact tissue (Fig. 1A); using STARmap, we were able sequencing of single-cell to identify organizational principles of a full spectrum of cell types, which would not have been transcriptional states otherwise accessible for identification in the adult mammalian brain. Xiao Wang1*, William E. Allen1,2*, Matthew A. Wright1,3, Emily L. Sylwestrak1, Nikolay Samusik4, Sam Vesuna1, Kathryn Evans1, Cindy Liu1, Charu Ramakrishnan1, Results Jia Liu5, Garry P. Nolan4†, Felice-Alessio Bava4†‡, Karl Deisseroth1,3,6† Design and validation of STARmap principles Retrieving high-content gene-expression information while retaining three-dimensional (3D) One component is an efficient approach for in positional anatomy at cellular resolution has been difficult, limiting integrative understanding of situ amplification of a library of cDNA probes structure and function in complex biological tissues. We developed and applied a technology hybridized with cellular RNAs (this approach Downloaded from for 3D intact-tissue RNA sequencing, termed STARmap (spatially-resolved transcript amplicon is termed SNAIL, for specific amplification of readout mapping), which integrates hydrogel-tissue chemistry, targeted signal amplification, nucleic acids via intramolecular ligation). Reverse and in situ sequencing.The capabilities of STARmap were tested by mapping 160 to 1020 genes transcription may be the major efficiency-limiting simultaneously in sections of mouse brain at single-cell resolution with high efficiency, step for in situ sequencing (7, 21), and SNAIL accuracy, and reproducibility. Moving to thick tissue blocks, we observed a molecularly defined bypasses this step with a pair of primer and gradient distribution of excitatory-neuron subtypes across cubic millimeter–scale volumes padlock probes (fig. S1A) designed so that only (>30,000 cells) and a short-range 3D self-clustering in many inhibitory-neuron subtypes that when both probes hybridize to the same RNA http://science.sciencemag.org/ couldbeidentifiedanddescribedwith3DSTARmap. molecule can the padlock probe be circularized and rolling-circle-amplified to generate a DNA nanoball (amplicon) that contains multiple copies n biological tissues, diversity of function in situ sequencing methods typically use signal ofthecDNAprobes(Fig.1,AtoD).Thismech- arises from diversity of form—in part, via the amplification, which is important for the detec- anism ensures target-specific signal amplification complexity of cell-specific gene expression, tion of short transcripts (such as neuropeptides) and excludes noise that invariably otherwise I which defines the distinct three-dimensional and for high-quality imaging in thick tissue arises from nonspecific hybridization of single (3D) molecular anatomy and cellular proper- blocks. However, current sequencing methods probes. The outcome includes much higher ab- ties of each tissue. In situ transcriptomic tools have not yet been successfully applied to 3D solute intensity and signal-to-noise ratio (SNR) for the spatial mapping of gene expression with volumes of intact tissue because of fundamental as compared with those of commercial single- subcellular resolution have emerged that may limitations in requisite sensitivity, fidelity, and molecule fluorescent in situ hybridization (smFISH) on January 3, 2020 be applicable to probing these tissue structure– scalability for throughput in tissues such as the probes (fig. S1, B to F)

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