Brief Communication

Massively parallel single-nucleus RNA-seq with DroNc-seq

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Abstract

Single-nucleus RNA sequencing (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but it does not provide high throughput. Here, we develop DroNc-seq: massively parallel sNuc-seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient, and unbiased classification of cell types, paving the way for systematic charting of cell atlases.

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Acknowledgements

We thank R. Macare, A. Rotem, C. Muus, and E. Drokhlyansky for helpful discussions, T. Habib for babysitting, T. Tickle and A. Bankapur for technical support, and L. Gaffney and A. Hupalowska for help with graphics. Work was supported by the Klarman Cell Observatory, National Institute of Mental Health (NIMH) grant U01MH105960, National Cancer Institute (NCI) grant 1R33CA202820-1 and NIAID grant U24AI118672-01 (to A.R.), and Koch Institute Support (core) grant P30-CA14051 from the NCI. Microfluidic devices were fabricated at the Center for Nanoscale Systems, Harvard University, supported by National Science Foundation award no. 1541959. N.H. is supported by HHMI through the HHWF, A.R. is supported by HHMI, and F.Z. is supported by the New York Stem Cell Foundation. F.Z. is supported by NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, HHMI, and the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations, and by J. and P. Poitras, R. Metcalfe, and D. Cheng. D.A.W. thanks NSF DMR-1420570, NSF DMR-1310266, and NIH P01HL120839 grants for their support. GTEx is supported by the NIH Common Fund (Contract HHSN268201000029C to K.A.).

Author information

Author notes

    • Anindita Basu

    Present address: Department of Medicine, University of Chicago, Chicago, Illinois, USA, and Center for Nanoscale Materials, Argonne National Laboratory, Lemont, Illinois, USA.

    • Naomi Habib
    • , Inbal Avraham-Davidi
    •  & Anindita Basu

    These authors contributed equally to this work.

Affiliations

  1. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Naomi Habib
    • , Inbal Avraham-Davidi
    • , Anindita Basu
    • , Tyler Burks
    • , Karthik Shekhar
    • , Matan Hofree
    • , Orit Rozenblatt-Rosen
    •  & Aviv Regev
  2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Naomi Habib
    • , Sourav R Choudhury
    • , François Aguet
    • , Ellen Gelfand
    • , Kristin Ardlie
    •  & Feng Zhang
  3. McGovern Institute, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Naomi Habib
    • , Sourav R Choudhury
    •  & Feng Zhang
  4. John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.

    • Anindita Basu
    •  & David A Weitz
  5. Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

    • David A Weitz
  6. Howard Hughes Medical Institute, Department of Biology, Koch Institute of Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Aviv Regev

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Contributions

N.H., I.A.D., A.B., O.R., F.Z., and A.R. conceived the study. A.R. and N.H. devised analyses. N.H., K.S., M.H., and F.A. analyzed the data. A.B. designed and fabricated the microfluidics device. D.A.W. devised the microfluidics design. A.B., I.A.D., N.H., and T.B. designed and conducted the experiments. S.R.C. provided mouse brain tissue. E.G. and K.A. provided human brain tissue. N.H., I.A.D., A.B., and A.R. wrote the paper with input from all of the authors.

Competing interests

N.H., A.B., I.A.D., D.A.W., F.Z., and A.R. are co-inventors on international patent application PCT/US16/59239 of Broad Institute, Harvard and MIT, relating to inventions of methods of this manuscript. A.R. is a member of Scientific Advisory Boards for Thermo Fisher Scientific, Syros Pharmaceuticals, and Driver Genomics.

Corresponding authors

Correspondence to Feng Zhang or Aviv Regev.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11.

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    Step-by-step protocol for DroNc-seq.

Excel files

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    Supplementary Table 1

    Species mixing.

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    Supplementary Table 2

    Cells Nuclei 3T3.

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    Supplementary Table 3

    Data Info Mouse.

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    Supplementary Table 4

    Mouse Cluster Markers.

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    Supplementary Table 5

    Mouse GABAergic Cluster Markers.

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    Supplementary Table 6

    Tissue Samples from GTEX.

  7. 7.

    Supplementary Table 7

    Data Info Human.

  8. 8.

    Supplementary Table 8

    Human Cluster Markers.

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    Supplementary Table 9

    Human GABAergic Clusters Markers.

Zip files

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    Supplementary Data 1

    CAD scheme of DroNc-seq microfluidic device.