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Nat Protoc DOI:10.1038/nprot.2016.090

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

Publication TypeJournal Article
Year of Publication2016
AuthorsAvraham, R, Haseley, N, Fan, A, Bloom-Ackermann, Z, Livny, J, Hung, DT
JournalNat Protoc
Volume11
Issue8
Pages1477-91
Date Published2016 Aug
ISSN1750-2799
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.

DOI10.1038/nprot.2016.090
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/27442864?dopt=Abstract

Alternate JournalNat Protoc
PubMed ID27442864