title: A Sample-to-Sequence Protocol for Genus Targeted Transcriptomic Profiling: Application to MarineSynechococcus.
abstract: Recent studies using whole community metagenomic and metatranscriptomic approaches are revealing important new insights into the functional potential and activity of natural marine microbial communities. Here, we complement these approaches by describing a complete ocean sample-to-sequence protocol, specifically designed to target a single bacterial genus for purposes of both DNA and RNA profiling using fluorescence activated cell sorting (FACS). The importance of defining and understanding the effects of a sampling protocol are critical if we are to gain meaningful data from environmental surveys. Rigorous pipeline trials with a cultured isolate,Synechococcussp. BL107 demonstrate that water filtration has a well-defined but limited impact on the transcriptomic profile of this organism, whilst sample storage and multiple rounds of cell sorting have almost no effect on the resulting RNA sequence profile. Attractively, the means to replicate the sampling strategy is within the budget and expertise of most researchers.
authors: School of Life Sciences, University of Warwick Coventry, UK.
authors: Frances D
authors: FD
authors: Pitt
authors: Warwick Medical School, University of Warwick Coventry, UK.
authors: Andrew
authors: A
authors: Millard
authors: Department of Biochemistry and Biomolecular Science, Faculty of Science and Engineering, Macquarie University Sydney, NSW, Australia.
authors: Martin
authors: M
authors: Ostrowski
authors: Sydney Cytometry, Centenary Institute, University of Sydney Sydney, NSW, Australia.
authors: Suat
authors: S
authors: Dervish
authors: Department of Biochemistry and Biomolecular Science, Faculty of Science and Engineering, Macquarie University Sydney, NSW, Australia.
authors: Sophie
authors: S
authors: Mazard
authors: Department of Biochemistry and Biomolecular Science, Faculty of Science and Engineering, Macquarie University Sydney, NSW, Australia.
authors: Ian T
authors: IT
authors: Paulsen
authors: National Oceanography Centre Southampton, UK.
authors: Mikhail V
authors: MV
authors: Zubkov
authors: School of Life Sciences, University of Warwick Coventry, UK.
authors: David J
authors: DJ
authors: Scanlan
keywords: RNA; Synechococcus; flow cytometry; fluorescence activated cell sorting; marine microbiology; transcriptomics