title: A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes.
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.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Roi
authors.initials: R
authors.lastname: Avraham
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Haseley
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Fan
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Zohar
authors.initials: Z
authors.lastname: Bloom-Ackermann
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.|Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Livny
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.|Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.|Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.|Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Deborah T
authors.initials: DT
authors.lastname: Hung
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D020869:Gene Expression Profiling / Q000379:methods*; D054884:Host-Pathogen Interactions; D057230:Limit of Detection; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D012329:RNA, Bacterial / Q000235:genetics*; D012475:Salmonella / Q000166:cytology / Q000235:genetics* / Q000502:physiology*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013997:Time Factors
chemical_list: D012329:RNA, Bacterial