The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. While RNA-Seq has greatly advanced our ability to study the transcriptomes of prokaryote and eukaryotes separately, limitations in existing protocols for generating and analyzing 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 to analyze both mammalian and microbial reads from mixed hostpathogen 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, suitable for large-scale studies, which will enable the field to obtain in-depth analysis of hostpathogen interactions in infection models
