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      Three-dimensional intact-tissue sequencing of single-cell transcriptional states

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          Abstract

          Retrieving high-content gene-expression information while retaining three-dimensional (3D) positional anatomy at cellular resolution has been difficult, limiting integrative understanding of structure and function in complex biological tissues. We developed and applied a technology for 3D intact-tissue RNA sequencing, termed STARmap (spatially-resolved transcript amplicon readout mapping), which integrates hydrogel-tissue chemistry, targeted signal amplification, and in situ sequencing. The capabilities of STARmap were tested by mapping 160 to 1020 genes simultaneously in sections of mouse brain at single-cell resolution with high efficiency, accuracy, and reproducibility. Moving to thick tissue blocks, we observed a molecularly defined gradient distribution of excitatory-neuron subtypes across cubic millimeter–scale volumes (>30,000 cells) and a short-range 3D self-clustering in many inhibitory-neuron subtypes that could be identified and described with 3D STARmap.

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          Author and article information

          Journal
          0404511
          7473
          Science
          Science
          Science (New York, N.Y.)
          0036-8075
          1095-9203
          18 December 2018
          21 June 2018
          27 July 2018
          21 January 2019
          : 361
          : 6400
          : eaat5691
          Affiliations
          [1 ]Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
          [2 ]Neuroscience Program, Stanford University, CA 94305, USA.
          [3 ]Department of Psychiatry and Behavioral Sciences, Stanford University, CA 94305, USA.
          [4 ]Baxter Laboratory, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.
          [5 ]Department of Chemical Engineering, Stanford University, CA 94305, USA.
          [6 ]Howard Hughes Medical Institute, Stanford University, CA 94305, USA.
          Author notes
          [‡]

          Present address: Institut Curie, PSL Research University, Université Paris Sud, Université Paris-Saclay, Centre Universitaire, CNRS UMR 3348, Orsay 91405, France.

          Author contributions: X.W. and K.D. initiated the STARmap project to integrate HTC with in situ sequencing. X.W. developed the STARmap HTC, SEDAL sequencing, and STARmap hardware, designed all the DNA probes, and conducted the experiments. W.E.A. developed the STARmap software pipeline and analyzed the sequencing data. N.S., F.-A.B., and G.P.N. designed the initial versions of the SNAIL system. X.W. and F.-A.B. developed the SNAIL process for mouse brain tissue and compared SNAIL with other in situ methods. M.A.W. and E.L.S. conducted animal behavior and preparation of mouse brain tissue and contributed valuable advice. M.A.W. compared Nissl staining with other segmentation methods. W.E.A. and E.L.S. designed the visual-stimulus and cocaine-stimulus procedures. S.V. generated the CLARITY data with PV transgenicmice. K.E. and C.R. contributed to preparation of cell cultures. C.L. assisted with experiments. J.L. assisted with experiments and graphic design. K.D. supervised all aspects of the work. X.W., W.E.A., and K.D. interpreted the STARmap data, designed and prepared the figures, and wrote the manuscript, with edits from all authors.

          Article
          PMC6339868 PMC6339868 6339868 hhmipa1001922
          10.1126/science.aat5691
          6339868
          29930089
          88db7884-2161-4bf9-90ab-8afec64e4471
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