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


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

Author information




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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