Avraham R, Haseley N, Fan A, Bloom-Ackermann Z, Livny J, Hung DT (2016) A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes. Nature Protocols 11: 8, 1477-1491. doi:10.1038/nprot.2016.090

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.

This study provides 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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