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A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

Nature Protocols volume 11, pages 1477–1491 (2016)Cite this article

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Abstract

The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen–host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host–pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host–pathogen interactions in infection models.

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Figure 1: Overview of the simultaneous host–pathogen RNA-seq analysis protocol.
Figure 2: Electropherogram of total RNA extracted from Salmonella-infected macrophages.
Figure 3: Electropherogram of the resulting RNA-seq libraries.

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Acknowledgements

This work was supported by an NIH grant (HG002295 to N.H.)

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Author notes

  1. Roi Avraham

    Present address: Present address: Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.,

Authors and Affiliations

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

    Roi Avraham, Nathan Haseley, Amy Fan, Zohar Bloom-Ackermann, Jonathan Livny & Deborah T Hung

  2. Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Jonathan Livny

  3. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Deborah T Hung

  4. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Deborah T Hung

  5. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Deborah T Hung

Authors
  1. Roi Avraham
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Contributions

R.A. designed the experiments. R.A., N.H., A.F. and Z.B.-A. conducted the experimental work. N.H. and J.L. performed the computational analysis. R.A., J.L. and D.T.H. wrote the manuscript.

Corresponding author

Correspondence to Deborah T Hung.

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The authors declare no competing financial interests.

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Avraham, R., Haseley, N., Fan, A. et al. A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes. Nat Protoc 11, 1477–1491 (2016). https://doi.org/10.1038/nprot.2016.090

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