An efficient field and laboratory workflow for plant phylotranscriptomic projects.
OBJECTIVE
We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation.


RESULTS
A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae.


CONCLUSIONS
Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10-12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds.
Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, 830 North University Avenue, Ann Arbor, Michigan 48109 USA.
Ya
Y
Yang
Department of Biology, Oberlin College, 119 Woodland Street, Oberlin, Ohio 44074-1097 USA.
Michael J
MJ
Moore
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
Samuel F
SF
Brockington
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
Alfonso
A
Timoneda
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
Tao
T
Feng
Department of Biological Sciences and Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, Idaho 83844 USA.
Hannah E
HE
Marx
Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, 830 North University Avenue, Ann Arbor, Michigan 48109 USA.
Joseph F
JF
Walker
Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, 830 North University Avenue, Ann Arbor, Michigan 48109 USA.
Stephen A
SA
Smith
Caryophyllales; RNA; cryogenic field sampling; phylogenomics; phylotranscriptomics