Abstract
Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell wall, which is a challenge for RNA purification. This protocol was designed specifically to breach the cell wall and isolate high-quality RNA suitable for RNA-Seq studies.
Keywords: Chromochloris zofingiensis, Algae, RNA purification, RNA-Seq, Cell wall
Background
Chromochloris zofingiensis is a small unicellular green alga from the chlorophyte lineage (Dönz, 1934). Previously, this species has been described in the literature with the genera Muriella, Chlorella, and Mychonastes (Fučíková and Lewis, 2012). There are strong economic interests in C. zofingiensis because it is capable of producing large quantities of lipids for biofuels and shows promise as a source of the commercially valuable nutraceutical astaxanthin (Breuer et al., 2012; Mulders et al., 2014; Liu et al., 2016). Recently, a high-quality, chromosome-level genome assembly and accompanying annotations were published, which facilitates systems level analyses like RNA-Seq (Roth et al., 2017). The following protocol was designed to produce highly purified total RNA, including miRNA, from liquid cultures of C. zofingiensis suitable for RNA-Seq. C. zofingiensis cells are protected by a robust cell wall, which this protocol was designed to penetrate. Starting material for the protocol can be up to 2.5 x 108 total cells. This protocol should yield at least 20 µg of highly purified RNA suitable for the preparation of RNA-Seq libraries by standard kits, such as the Illumina TruSeq Stranded Total RNA kit.
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
RNA is extremely sensitive to damage by RNase. Careful laboratory hygiene is crucial for minimizing this risk. Additionally, cells must be kept frozen until lysis buffer is added to prevent RNA degradation. When preparing many RNA samples, some may not satisfy the quality control criteria specified in the Data analysis section. It may be prudent to prepare more replicate samples than your experiment requires so that some may be discarded at the end of this protocol without compromising the overall study.
Recipes
Acknowledgments
This protocol has been adapted from a previously published paper (Roth et al., 2017). This work was supported by the Agriculture and Food Research Initiative Competitive Grant No. 2013-67012-21272 from the USDA National Institute of Food and Agriculture (to M.S.R), and by a cooperative agreement with the US Department of Energy Office of Science, Office of Biological and Environmental Research program under Award DE-FC02-02ER63421. The authors declare no conflicts of interest or competing interests.
References
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