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Expansion Sequencing of RNA Barcoded Neurons in the Mammalian Brain: Progress and Implications for Molecularly Annotated Connectomics

Daniel R. Goodwin, Alex Vaughan, Daniel Leible, Shahar Alon, Gilbert L. Henry, Anne Cheng, Xiaoyin Chen, Ruihan Zhang, Andrew G. Xue, Asmamaw T. Wassie, Anubhav Sinha, Yosuke Bando, Atsushi Kajita, Adam H. Marblestone, Anthony M. Zador, Edward S. Boyden, George M. Church, View ORCID ProfileRichie E. Kohman
doi: https://doi.org/10.1101/2022.07.31.502046
Daniel R. Goodwin
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
2McGovern Institute, MIT, Cambridge, MA, USA
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Alex Vaughan
3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
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Daniel Leible
2McGovern Institute, MIT, Cambridge, MA, USA
4Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
5Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA
6Picower Institute for Learning and Memory, MIT, Cambridge, MA, USA
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Shahar Alon
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
2McGovern Institute, MIT, Cambridge, MA, USA
7Faculty of Engineering, Gonda Brain Research Center and Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel
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Gilbert L. Henry
3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
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Anne Cheng
4Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Xiaoyin Chen
3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
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Ruihan Zhang
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
2McGovern Institute, MIT, Cambridge, MA, USA
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Andrew G. Xue
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
2McGovern Institute, MIT, Cambridge, MA, USA
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Asmamaw T. Wassie
2McGovern Institute, MIT, Cambridge, MA, USA
8Department of Biological Engineering, MIT, Cambridge, MA, USA
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Anubhav Sinha
9Harvard-MIT Program in Health Sciences and Technology, MIT, Cambridge, MA, USA
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Yosuke Bando
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
10Kioxia Corporation, Minato-ku, Tokyo, Japan
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Atsushi Kajita
11Fixstars Solutions Inc, Irvine, CA, USA
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Adam H. Marblestone
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
12Federation of American Scientists, Washington, DC, USA
13Convergent Research, Cambridge, MA, USA
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Anthony M. Zador
3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
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Edward S. Boyden
1Department of Media Arts and Sciences, MIT, Cambridge, MA, USA
2McGovern Institute, MIT, Cambridge, MA, USA
5Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA
8Department of Biological Engineering, MIT, Cambridge, MA, USA
14Koch Institute for Integrative Cancer Research, Department of Biology, MIT, Cambridge, MA, USA
15Howard Hughes Medical Institute, Chevy Chase, MD, USA
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George M. Church
4Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
16Department of Genetics, Harvard Medical School, Boston, MA, USA
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Richie E. Kohman
2McGovern Institute, MIT, Cambridge, MA, USA
4Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
16Department of Genetics, Harvard Medical School, Boston, MA, USA
17Wyss Center for Bio and Neuroengineering, Geneva, Switzerland
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  • ORCID record for Richie E. Kohman
  • For correspondence: richie.kohman@wysscenter.ch
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Abstract

Mapping and molecularly annotating mammalian neural circuits is challenging due to the inability to uniquely label cells while also resolving subcellular features such as synaptic proteins or fine cellular processes. We argue that an ideal technology for connectomics would have the following characteristics: the capacity for robust distance-independent labeling, synaptic resolution, molecular interrogation, and scalable computational methods. The recent development of high-diversity cellular barcoding with RNA has provided a way to overcome the labeling limitations associated with spectral dyes, however performing all-optical circuit mapping has not been demonstrated because no method exists to image barcodes throughout cells at synaptic-resolution. Here we show ExBarSeq, an integrated method combining in situ sequencing of RNA barcodes, immunostaining, and Expansion Microscopy coupled with an end-to-end software pipeline that automatically extracts barcode identities from large imaging datasets without data processing bottlenecks. As a proof of concept, we applied ExBarSeq to thick tissue sections from mice virally infected with MAPseq viral vectors and demonstrated the extraction of 50 barcoded cells in the visual cortex as well as cell morphologies uncovered via immunostaining. The current work demonstrates high resolution multiplexing of exogenous barcodes and endogenous synaptic proteins and outlines a roadmap for molecularly annotated connectomics at a brain-wide scale.

Competing Interest Statement

A.M.Z. consults for and is a founder of Cajal Neuroscience. E.S.B. is an inventor on multiple patents relating to ExM, and he is a cofounder of Expansion Technologies, which has commercial interests in the space of ExM. G.M.C. is a cofounder and SAB member of ReadCoor and is an adviser to 10x Genomics after their acquisition of ReadCoor. Conflict of interest link for G.M.C.: http://arep.med.harvard.edu/gmc/tech.html. R.E.K. was a founding scientist of and has equity in Expansion Technologies.

Footnotes

  • https://github.com/dgoodwin208/exbarseq

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Expansion Sequencing of RNA Barcoded Neurons in the Mammalian Brain: Progress and Implications for Molecularly Annotated Connectomics
Daniel R. Goodwin, Alex Vaughan, Daniel Leible, Shahar Alon, Gilbert L. Henry, Anne Cheng, Xiaoyin Chen, Ruihan Zhang, Andrew G. Xue, Asmamaw T. Wassie, Anubhav Sinha, Yosuke Bando, Atsushi Kajita, Adam H. Marblestone, Anthony M. Zador, Edward S. Boyden, George M. Church, Richie E. Kohman
bioRxiv 2022.07.31.502046; doi: https://doi.org/10.1101/2022.07.31.502046
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Expansion Sequencing of RNA Barcoded Neurons in the Mammalian Brain: Progress and Implications for Molecularly Annotated Connectomics
Daniel R. Goodwin, Alex Vaughan, Daniel Leible, Shahar Alon, Gilbert L. Henry, Anne Cheng, Xiaoyin Chen, Ruihan Zhang, Andrew G. Xue, Asmamaw T. Wassie, Anubhav Sinha, Yosuke Bando, Atsushi Kajita, Adam H. Marblestone, Anthony M. Zador, Edward S. Boyden, George M. Church, Richie E. Kohman
bioRxiv 2022.07.31.502046; doi: https://doi.org/10.1101/2022.07.31.502046

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