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Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput

Nature Methods volume 14, pages 395398 (2017) | Download Citation

  • An Erratum to this article was published on 29 June 2017

This article has been updated

Abstract

Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.

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Change history

  • 21 June 2017

    The doi, link, and publication date of the Seq-Well protocol in the Protocol Exchange were updated; the doi and link have been corrected from http://dx.doi.org/10.1038/protex.2017.006 to http://dx.doi.org/10.1038/protex.2017.006a, and the date has been changed from 2016 to 2017. The grant number U24 AI11862 was incorrect; it has been corrected to U24 AI118762. These errors have been updated in the HTML and PDF versions of the article.

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Acknowledgements

We thank K. Shekhar, T. Tickle, and M. Xie for fruitful discussions. This work was supported by the Searle Scholars Program (A.K.S.), the Beckman Young Investigator Program (NIH, A.K.S.), NIH New Innovator Award DP2 OD020839 (A.K.S.), NIH U24 AI118762 (A.K.S.), P50 HG006193 (A.K.S.), the Bill and Melinda Gates Foundation grant 03629000189 (A.K.S., J.C.L. and S.F.), the Ragon Institute (A.K.S. and S.F.), the Burroughs Wellcome Foundation (S.F.), NIH P30 AI060354 (S.F.), DP3 DK09768101 (NIH, J.C.L.), P01 AI045757 (J.C.L.), NIH R21 AI106025 (J.C.L.), NIH R56 AI104274 (J.C.L.), the W.M. Keck Foundation (J.C.L.), and the US Army Research Office through the Institute for Soldier Nanotechnologies, under contract number W911NF-13-D-0001 (J.C.L.). This work was also supported in part by the Koch Institute Support (core) NIH Grant P30-CA14051 from the National Cancer Institute. J.C.L. is a Camille Dreyfus Teacher–Scholar.

Author information

Author notes

    • Todd M Gierahn
    • , Marc H Wadsworth II
    •  & Travis K Hughes

    These authors contributed equally to this work.

    • J Christopher Love
    •  & Alex K Shalek

    These senior authors contributed equally to this work.

Affiliations

  1. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, USA.

    • Todd M Gierahn
    •  & J Christopher Love
  2. Institute for Medical Engineering & Science (IMES) and Department of Chemistry, MIT, Cambridge, Massachusetts, USA.

    • Marc H Wadsworth II
    • , Travis K Hughes
    •  & Alex K Shalek
  3. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Marc H Wadsworth II
    • , Travis K Hughes
    • , J Christopher Love
    •  & Alex K Shalek
  4. Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA.

    • Marc H Wadsworth II
    • , Travis K Hughes
    • , Bryan D Bryson
    • , Sarah Fortune
    • , J Christopher Love
    •  & Alex K Shalek
  5. Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA.

    • Bryan D Bryson
    •  & Sarah Fortune
  6. Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York, USA.

    • Andrew Butler
    •  & Rahul Satija
  7. New York Genome Center, New York, New York, USA.

    • Andrew Butler
    •  & Rahul Satija

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Contributions

T.M.G., M.H.W. II, T.K.H., J.C.L. and A.K.S. developed the concepts and designed the study. T.M.G., M.H.W. II, T.K.H. and B.D.B. performed the experiments. T.M.G., M.H.W., T.K.H., A.B. and R.S. performed bioinformatic analysis. S.F. helped design and interpret the M. tuberculosis experiments. All authors analyzed and interpreted the data. T.M.G., M.H.W. II, T.K.H., J.C.L. and A.K.S. wrote the manuscript with feedback from all authors.

Competing interests

T.M.G., M.H.W. II, T.K.H., J.C.L., A.K.S., and the Broad Institute and the Massachusetts Institute of Technology have filed a patent application (patent no. PCT/US17/13791) that relates to Seq-Well, compositions of matter, the outlined experimental and computational methods and uses thereof.

Corresponding authors

Correspondence to J Christopher Love or Alex K Shalek.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Tables 1–2

  2. 2.

    Supplementary Protocol

    Seq-Well protocol

Zip files

  1. 1.

    Supplementary Table 3

    Gene Expression Matrix for PBMCs

  2. 2.

    Supplementary Table 5

    Gene Expression Matrix for Mtb-Exposed Monocyte-Derived Macrophages and Unexposed Control Cells

Excel files

  1. 1.

    Supplementary Table 4

    PBMC Cluster Enrichments

  2. 2.

    Supplementary Table 6

    TB Cluster Enrichments

  3. 3.

    Supplementary Table 7

    Cluster Enrichments between Exposure Groups

  4. 4.

    Supplementary Table 8

    Differentially Expressed Genes between TB Exposed and Unexposed Cells within Each Cluster

  5. 5.

    Supplementary Table 9

    TB Infection by Cluster Enrichments

  6. 6.

    Supplementary Table 10

    GSEA Comparisons of Exposed and Unexposed Cells within Each Cluster

Videos

  1. 1.

    Bead Loading

    Video demonstration of bead loading on microwells arrays.

  2. 2.

    Membrane Attachment

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DOI

https://doi.org/10.1038/nmeth.4179

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