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Authors: Jeric Harper
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab

Proper citation: Jeric Harper 2017. Diatom Transformation via Bacterial Conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.hgpb3vn Copy   

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Authors: Tamara Matute, Isaac Nuñez, Peter Von Dassow, Fernan Federici
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Laboratorio de Tecnologias Libres

Proper citation: Tamara Matute, Isaac Nuñez, Peter Von Dassow, Fernan Federici 2017. Fabrication of DNA constructs by Gibson Assembly and Golden Gate reactions. protocols.io https://dx.doi.org/10.17504/protocols.io.hpib5ke Copy   

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Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab

Proper citation: Maria Rubio-Brotons 2016. Staining protocol for unicellular protists: Lysotracker and Dapi. protocols.io https://dx.doi.org/10.17504/protocols.io.gg4btyw Copy   

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Authors: Marie D., Le Gall F., Edern R., Gourvil P., Vaulot, D.
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G), Roscoff Culture Collection
Summary: This protocol can be used :to isolate novel cultures from natural samplesto isolate novel cultures from enriched samplesto purify existing cultures and remove contaminantsto obtain clonal cultures from a unialgal strainReference : Marie, D., Le Gall, F., Edern, R., Gourvil, P. & Vaulot, D. 2017. Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry. J. Phycol. in press.

Proper citation: Marie D., Le Gall F., Edern R., Gourvil P., Vaulot, D. 2017. Establishment of clonal algal cultures by flow cytometry sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.hdib24e Copy   

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Authors: Lawrence A. Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This procedure was developed to investigate the possible use of RNAi through bacterial feeding as a means of knocking down expression of particular genes.While the utility of RNAi is still being investigated, the method is effective in growing Euplotes crassus to higher densities than is possible using algae alone. Using moderately dense cultures of algae, we typically achieve cell densities of 500-1,000 cells/ml, while with the E. coli supplemented cultures we describe here, Euplotes cell densities exceed 3,000 cells/ml. In fact, preliminary studies mixing a volume of a Euplotes culture with an equal volume of resuspended E. coli have produced a density of ~8,000 cells/ml.While the described procedure involves small cultures, it can likely be scaled up as required.For some species of Euplotes, long-term culture using bacteria as a food source has not been successful (C. Miceli, personal communication), suggesting that some essential nutrient is not being provided in sufficient quantitities

Proper citation: Lawrence A. Klobutcher 2017. Culturing Euplotes crassus to high densities using a combination of algae and bacteria as the food source.. protocols.io https://dx.doi.org/10.17504/protocols.io.gw2bxge Copy   

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Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using Lipofectamine 2000 as vehicle. protocols.io https://dx.doi.org/10.17504/protocols.io.g42byye Copy   

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Authors: José A. Fernández Robledo
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: José A. Fernández Robledo Nastasia J. Freyria Bigelow Laboratory for Ocean Sciences

Proper citation: José A. Fernández Robledo 2019. Transfection of Crypthecodinium cohnii using labelled DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.z26f8he Copy   

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Authors: Amanda Scholes
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: A protocol for making multiplexed RNA-seq libraries using KAPA Biosystems mRNA HyperPrep Kit, optimized for Saccharomyces cerevisiae.

Proper citation: Amanda Scholes 2019. RNA-seq Library Construction with the KAPA mRNA HyperPrep Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.uueewte Copy   

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Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2017. Quick staining procedure of nuclei in Euplotes using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.hjfb4jn Copy   

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Authors: Pavel Doležal and Vladimir Hampl
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: In this protocol we publish the maps and sequences of specific plasmids designed for the transformation of Bigelowiella natans, Pyramimonas parkeae, Eutreptiella gymnastica, Chromera velia and Pseudonitzchia delicatissima. All these plasmids contain gene for firefly lucipherase as a reporter, some of them contain PAC (puromycin) or G418 (neomycin) resistance gene. All genes are flanked by organism specific up- and downstream gene regions extracted from genomic and transcriptomic data. It should be noted that we were not successful in any transformation by these plasmids, but it does not mean that the problem is in the plasmids. All plasmids are available upon request. The plasmid maps and sequences can be downloaded as a word file from More/Material section of this protocol. 

Proper citation: Pavel Doležal and Vladimir Hampl 2017. Plasmids for transformation of Bigelowiella, Pyramimonas, Eutreptiella and Chromera. protocols.io https://dx.doi.org/10.17504/protocols.io.hh6b39e Copy   

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Authors: Eric Gann
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: We have developed a plaque assay for AaV with a modified protocol from Schroeder et. al 2002. Protocol modified from:Schroeder, D.C., et al., Coccolithovirus (Phycodnaviridae): Characterisation of a new large dsDNAalgal virus that infects Emiliania huxleyi. Archives of Virology, 2002.147(9): p. 1685-1698.

Proper citation: Eric Gann 2017. Plaque Assay for the Aureococcus anophagefferens Virus (AaV). protocols.io https://dx.doi.org/10.17504/protocols.io.g2nbyde Copy   

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Authors: Margaret Brisbin, Lisa Mesrop
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Marine protists are important in all marine ecosystems, but many are not culturable and therefore difficult to study. The increasing development of single-cell techniques is allowing for more marine protists to be studied, but many single-cell protocols are only optimized for model organisms and do not perform well with marine plankton. The SMART-seq v4 manual specifically states that cells entering the pipeline should be washed and resuspended in cell culture-grade PBS, but most marine protists do not tolerate PBS. Additionally, many marine protists have mineral skeletons, the components of which may interfere with the chemistry of the SMART-seq kit. Calcium and Strontium both dissolve to form divalent cations which could saturate the kit's RNase inhibitor. When we synthesized cDNA directly from acantharians (SrSO4 skeletons) without first extracting RNA, the cDNA was highly degraded with a 300 bp average fragment length. Therefore, it is desirable to perform RNA extractions from single marine protists before cDNA synthesis and amplification, but the amount of RNA in single marine protists is too low to be extracted with column-based kits (even the MN Nucleospin RNA XS kit suggested in the SMART-seq manual). To overcome these problems, we developed a column-free method for extracting RNA from single marine protists by making use of Agencourt RNAClean XP magnetic beads. When this protocol is followed, the synthesized cDNA is higher quality with a 2,000 bp average fragment length. 

Proper citation: Margaret Brisbin, Lisa Mesrop 2018. Single-cell RNA extraction and cDNA library preparation from challenging marine protists (e.g. Acantharea). protocols.io https://dx.doi.org/10.17504/protocols.io.mvhc636 Copy   

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Authors: Mariana Rius
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Mariana Rius 2017. Cryopreservation of thraustochytrids. protocols.io https://dx.doi.org/10.17504/protocols.io.h9fb93n Copy   

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Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using FuGene HD Transfection Reagent as vehicle. protocols.io https://dx.doi.org/10.17504/protocols.io.g5bby2n Copy   

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Authors: Lisa Sudek, Alexandra Worden, Manny Ares
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Worden Lab
Summary: This protocol describes the transformation of the picoprasinophyte alga Micromonas commoda, a green alga that is found in tropical to temperate oceans. The strain used was genome-sequenced in (1). The original strain name was NOUM17, housed as RCC299. The latter was rendered axenic, genome-sequenced, and deposited at the CCMP (now NCMA) as CCMP2709 and also maintained in the Worden lab. The below protocol is an adaptation of transformation methods used for the choanoflagellate Salpingoeca rosetta. We are grateful to N. King and D. Booth for their invaluable support in initial stages of experiments, as well as help from C. Poirier, M. Hamilton, K. Hoadley, C. Eckmann and especially C. Yung throughout the entire development.

Proper citation: Lisa Sudek, Alexandra Worden, Manny Ares 2019. Transfection of Micromonas commoda CCMP2709. protocols.io https://dx.doi.org/10.17504/protocols.io.57kg9kw Copy   

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Authors: Deepak Nanjappa and Kathryn Coyne
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol was developed for the glass bead-mediated transformation of Heterosigma akashiwo. This species does not grow on solid medium, so transformants are grown "in bulk" and should not be considered clonal. We have repeated this protocol with success and have been able to maintain transformants on selection medium for several months. 

Proper citation: Deepak Nanjappa and Kathryn Coyne 2017. Glass bead transformation of Heterosigma akashiwo. protocols.io https://dx.doi.org/10.17504/protocols.io.hjkb4kw Copy   

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Authors: Alyssa Alsante
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Van Etten, J. (n.d.). Formulation of Modified Bold's Basal Medium (MBBM). Retrieved from http://ncv.unl.edu/vanettenlab/

Proper citation: Alyssa Alsante 2016. MBBM Media. protocols.io https://dx.doi.org/10.17504/protocols.io.gdrbs56 Copy   

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Authors: Marie D., Le Gall F., Edern R., Gourvil P., Vaulot, D.
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G), Roscoff Culture Collection
Summary: This protocol can be used :to isolate novel cultures from natural samplesto isolate novel cultures from enriched samplesto purify existing cultures and remove contaminantsto obtain clonal cultures from a unialgal strainReference : Marie, D., Le Gall, F., Edern, R., Gourvil, P. & Vaulot, D. 2017. Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry. J. Phycol. in press.

Proper citation: Marie D., Le Gall F., Edern R., Gourvil P., Vaulot, D. 2018. Establishment of clonal algal cultures by flow cytometry sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.mn4c5gw Copy   

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Authors: François-Yves Bouget, Valérie Vergé and Jean-Claude Lozano
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the preparation of cells and introduction of DNA into the cells by electroporation.  For selection of stable transformants or measure of transient gene expression see related protocols.

Proper citation: François-Yves Bouget, Valérie Vergé and Jean-Claude Lozano 2017. Transient transformation of Ostreococcus species (OTTH595, RCC809 and RCC802) and Bathycoccus. protocols.io https://dx.doi.org/10.17504/protocols.io.g86bzze Copy   

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Authors: Imen Lassadi
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Imen Lassadi 2017. Fluorescence activated cell sorting (FACS) of Perkinsus marinus transformants. protocols.io https://dx.doi.org/10.17504/protocols.io.hh2b38e Copy   

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