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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Temperature-driven, single-tube extractions of total RNA from mammalian cells, tissues, insects, bacteria and virus
This powerful, broad-specificity approach is ideal for gene expression from a very small number of cells, such as exosomes or single cells, without the need for harsh chemicals or further purification.