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Transfection of Salpingoeca rosetta Resource Report Resource Website |
David S. Booth, Heather Middleton, and Nicole King | DOI:10.17504/protocols.io.h8bb9sn | King Lab, Protist Research to Optimize Tools in Genetics (PROT-G) | This collection of protocols describes the preparation of reagents for transfecting DNA into the choanoflagellate Salpingoeca rosetta using a Nucleofector 4d Device and SF kit from Lonza. Plasmids that use S. rosetta promoters and regulatory elements to drive the expression of luciferase and fluorescent protein reporters are being deposited at Addgene. Acknowlegements: Arielle Woznica and Laura Wetzel for meticulous editing of protocol and clarifying steps in the procedure. The Gordon and Betty Moore Foundation for supporting this research. | HHMI/University of California, Berkeley, HHMI/University of California, Berkeley, HHMI/University of California, Berkeley | 1 | 2018 | David S. Booth, Heather Middleton, and Nicole King 2018. Transfection of Salpingoeca rosetta. protocols.io https://dx.doi.org/10.17504/protocols.io.h8bb9sn | 2021-04-15 09:15:30 | |||
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Euplotes crassus GFP-neo artificial nanochromosomes sequence Resource Report Resource Website |
Angela Piersanti, Rachele Cesaroni | DOI:10.17504/protocols.io.2atgaen | Protist Research to Optimize Tools in Genetics (PROT-G) | Euplotes crassus GFP-neo artificial nanochromosomes | University of Camerino, Universität Bern | 1 | 2019 | Angela Piersanti, Rachele Cesaroni 2019. Euplotes crassus GFP-neo artificial nanochromosomes sequence. protocols.io https://dx.doi.org/10.17504/protocols.io.2atgaen | 2021-04-15 09:15:29 | |||
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Small-scale silencing experiment in vegetative Euplotes crassus (provisional) Resource Report Resource Website |
Rachele Cesaroni | DOI:10.17504/protocols.io.hi5b4g6 | Protist Research to Optimize Tools in Genetics (PROT-G) | Universität Bern | 4 | 2017 | Rachele Cesaroni 2017. Small-scale silencing experiment in vegetative Euplotes crassus (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.hi5b4g6 | 2021-04-15 09:15:35 | ||||
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In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386) Resource Report Resource Website |
Binnypreet Kaur | DOI:10.17504/protocols.io.rmud46w | Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes | Cas9 Nuclease, S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA). | 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic | 1 | 2018 | Binnypreet Kaur 2018. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386). protocols.io https://dx.doi.org/10.17504/protocols.io.rmud46w | 2021-04-15 09:15:38 | |||
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Labyrinthulomycete DNA extraction protocol Resource Report Resource Website |
Jackie L. Collier | DOI:10.17504/protocols.io.n83dhyn | Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab | Modified from Lippmeier et al. 2009 | Stony Brook University | 1 | 2018 | Jackie L. Collier 2018. Labyrinthulomycete DNA extraction protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n83dhyn | 2021-04-15 09:15:38 | |||
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Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries Resource Report Resource Website |
April Woods, G Jason Smith | DOI:10.17504/protocols.io.gj6bure | Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab | This is a modified version of 'Conjugation of Thalassiosira pseudonana', publishedby J. Turnsek dx.doi.org/10.17504/protocols.io.f55bq86 .The protocol was modified to enhance viability in the diatom Pseudo-nitzschia multiseries. This version was used to transform Pseudo-nitzschia multiseries isolate 15091C3 with an episomal plasmid derived from pPtPUC3 to express a egfp gene under control of the P. multiseries actin promoter and termination domains. | Moss Landing Marine Labs, Moss Landing Marine Labs | 1 | 2017 | April Woods, G Jason Smith 2017. Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries. protocols.io https://dx.doi.org/10.17504/protocols.io.gj6bure | 2021-04-15 09:15:44 | |||
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TruSeq RNA Preparation with Modification for Four Rounds of Poly-A Selection Resource Report Resource Website |
Dr. Daniel Richter | DOI:10.17504/protocols.io.imucc6w | Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G) | March, 2012; based on sample preparation guide v2, catalog # RS-930-1021, part # 15026495 Rev. A, August 2011Modifications: samples in PCR tubes (because a plate/seal will not be used, some numbered steps are skipped); include two rounds of poly-A selection (with two separate sets of beads); repeat clean-up step after PCR to remove additional adapters; elute with 1.5 ul less volume off of beads (to leave 1 ul behind, not 2.5 ul) | Richter, Daniel J and Fozouni, Parinaz and Eisen, Michael and King, Nicole. Gene family innovation, conservation and loss on the animal stem lineage. 2018;7:e34226 https://doi.org/10.7554/eLife.34226 | https://elifesciences.org/articles/34226 | 1 | 2017 | Dr. Daniel Richter 2017. TruSeq RNA Preparation with Modification for Four Rounds of Poly-A Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.imucc6w | 2021-04-15 09:15:39 | ||
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Transfection of Diplonemids using AMAXA Nucleofactor apparatus Resource Report Resource Website |
Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2 | DOI:10.17504/protocols.io.hf2b3qe | Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes | 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic, 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic, 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic, 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic, 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic, 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005, České Budějovice, Czech Republic 2 Faculty of Sciences, University of South Bohemia, 37005, České Budějovice, Czech Republic | 1 | 2019 | Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2 2019. Transfection of Diplonemids using AMAXA Nucleofactor apparatus. protocols.io https://dx.doi.org/10.17504/protocols.io.hf2b3qe | 2021-04-15 09:15:42 | ||||
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Quick staining procedure of nuclei in Euplotes crassus using DAPI Resource Report Resource Website |
Rachele Cesaroni | DOI:10.17504/protocols.io.g3tbynn | Protist Research to Optimize Tools in Genetics (PROT-G) | 1 | 2017 | Rachele Cesaroni 2017. Quick staining procedure of nuclei in Euplotes crassus using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.g3tbynn | 2021-04-15 09:15:44 | |||||
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Trace Metal Solution for ESAW Media Resource Report Resource Website |
Ashley Humphrey | DOI:10.17504/protocols.io.gdabs2e | Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs) | Trace metals solution to be used in ESAW Media for Marine Phytoplankton | University of Tennessee, Knoxville | 1 | 2016 | Ashley Humphrey 2016. Trace Metal Solution for ESAW Media. protocols.io https://dx.doi.org/10.17504/protocols.io.gdabs2e | 2021-04-15 09:15:45 | |||
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Electroporation of Caecitellus sp. with FITC-dextran Resource Report Resource Website |
Nick Irwin, Elisabeth Hehenberger, Patrick Keeling | DOI:10.17504/protocols.io.35kgq4w | Protist Research to Optimize Tools in Genetics (PROT-G) | Caecitellus sp. is a small phagotroph belonging to the biocosoecid order of the stramenopiles. Caecitellus is found across global oceans but it's role in marine ecosystems, as with many heterotrophic flagellates, is unclear. We isolated a strain of Caecitellus (small subunit rRNA gene was 99.53% identical to Caecitellus parvulus and Caecitellus paraparvulus) from a contaminated culture of Oxyrrhis marina and assessed its amenability to transformation. Fluorescein isothiocyanate conjugated to dextran was successfully introduced into Caecitellus sp. using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). Cell viability was confirmed by observing motility and fluorescence 24 h after electroporation (Figure 1). Lower voltage pulses did not result in FITC uptake. Estimates of transformation effifiency were difficult to obtain due to the small size of the cells.Unfortunately, the isolated strain of Caecitellus perished in an incubator malfunction. However, these results may be transferable to Caecitellus parvulus, a model for heterotrophic flagellates. | University of British Columbia, University of British Columbia, University of British Columbia | 1 | 2019 | Nick Irwin, Elisabeth Hehenberger, Patrick Keeling 2019. Electroporation of Caecitellus sp. with FITC-dextran. protocols.io https://dx.doi.org/10.17504/protocols.io.35kgq4w | 2021-04-15 09:15:48 | |||
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Dispersing Labyrinthulomycete Cell Aggregates by Sonication Resource Report Resource Website |
Mariana Rius, Kashyapa Bandaralage | DOI:10.17504/protocols.io.hg2b3ye | Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab | Sonication procedure implemented to reduce labyrinthulomycete cell aggregation from culture for subsequent growth analyses. | Stony Brook University, Stony Brook University | 1 | 2017 | Mariana Rius, Kashyapa Bandaralage 2017. Dispersing Labyrinthulomycete Cell Aggregates by Sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.hg2b3ye | 2021-04-15 09:15:46 | |||
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Long staining procedure of nuclei in Euplotes crassus using DAPI Resource Report Resource Website |
Rachele Cesaroni | DOI:10.17504/protocols.io.2akgacw | Protist Research to Optimize Tools in Genetics (PROT-G) | Universität Bern | 1 | 2019 | Rachele Cesaroni 2019. Long staining procedure of nuclei in Euplotes crassus using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.2akgacw | 2021-04-15 09:15:50 | ||||
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Pour plating of Thalassiosira pseudonana (Tp) Resource Report Resource Website |
Jernej Turnsek, Chris Dupont | DOI:10.17504/protocols.io.i6qchdw | Protist Research to Optimize Tools in Genetics (PROT-G) | Here we present an efficient way to grow Tp colonies inside a 0.25% superclean agar matrix. This protocol could possibly be applicable to other marine microeukaryotes that are problematic to grow on fully solid support. | University of California, Berkeley, J. Craig Venter Institute | 2 | 2017 | Jernej Turnsek, Chris Dupont 2017. Pour plating of Thalassiosira pseudonana (Tp). protocols.io https://dx.doi.org/10.17504/protocols.io.i6qchdw | 2021-04-15 09:15:55 | |||
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Transient transfection of unicellular relative to animals Corallochytrium limacisporum with electroporation using Neon SystemVersion 1 Resource Report Resource Website |
Maria Rubio-Brotons | DOI:10.17504/protocols.io.hmwb47e | Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab | IBE CSIC-UPF | 1 | 2017 | Maria Rubio-Brotons 2017. Transient transfection of unicellular relative to animals Corallochytrium limacisporum with electroporation using Neon SystemVersion 1. protocols.io https://dx.doi.org/10.17504/protocols.io.hmwb47e | 2021-04-15 09:15:52 | ||||
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Transfection of Naegleria gruberi Resource Report Resource Website |
Anastasios Tsaousis | DOI:10.17504/protocols.io.hpub5nw | Protist Research to Optimize Tools in Genetics (PROT-G) | Faktorová D, Nisbet RER, Robledo JAF, Casacuberta E, Sudek L, Allen AE, Ares M, Aresté C, Balestreri C, Barbrook AC, Beardslee P, Bender S, Booth DS, Bouget F, Bowler C, Breglia SA, Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V, Freyria NJ, Fukuda K, García PA, Girguis PR, Gomaa F, Gornik SG, Guo J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A, Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibañez J, Irwin NAT, Ishii Y, Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi I, Li Z, Lin S, Lozano J, Luan F, Maruyama S, Matute T, Miceli C, Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, Vanclová AMGN, Nowacki M, Nuñez I, Pain A, Piersanti A, Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Smith GJ, Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, Turnšek J, Valach M, Vergé V, Dassow Pv, Haar Tvd, Waller RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, Lukeš J, Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nature Methods 17(5). doi: 10.1038/s41592-020-0796-x | University of Kent | https://doi.org/10.1038/s41592-020-0796-x | 1 | 2017 | Anastasios Tsaousis 2017. Transfection of Naegleria gruberi. protocols.io https://dx.doi.org/10.17504/protocols.io.hpub5nw | 2021-04-15 09:15:53 | ||
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Rolling circle amplification-mediated hairpin (hp)-RNA libraries Resource Report Resource Website |
Philip D. Weyman | DOI:10.17504/protocols.io.hnrb5d6 | Protist Research to Optimize Tools in Genetics (PROT-G) | A sequence-independent method to turn genomic DNA into libraries of hairpins. An efficient way to get a different adapter on each end of a DNA fragment is to clone it and digest the fragment back out. Design the vector to have unique restriction sites outside the unique regions you want to put on each end. After digestion, your DNA fragments will have two different "adapters" on each end.Adapted from: Wang L., Zheng J., Luo Y., Xu T., Zhang Q., Zhang L., Xu M., Wan J., Wang MB., Zhang C., Fan Y. 2013. Construction of a genomewide RNAi mutant library in rice. Plant Biotechnology Journal 11:997–1005. DOI: 10.1111/pbi.12093. | J. Craig Venter Institute | 1 | 2017 | Philip D. Weyman 2017. Rolling circle amplification-mediated hairpin (hp)-RNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.hnrb5d6 | 2021-04-15 09:15:54 | |||
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Nucleofection of Pyramimonas parkeae, Chromera velia, Bigellowiella natans, Eutreptiella gymnastica, Neovalkampfia damariscottae Resource Report Resource Website |
Natalia Wandyszewska and Vladimir Hampl | DOI:10.17504/protocols.io.ibucanw | Protist Research to Optimize Tools in Genetics (PROT-G) | The protocol decribes the procedure and results of our attempts to transform several species of marine protists by nucleofection. | Charles University in Prague | 1 | 2017 | Natalia Wandyszewska and Vladimir Hampl 2017. Nucleofection of Pyramimonas parkeae, Chromera velia, Bigellowiella natans, Eutreptiella gymnastica, Neovalkampfia damariscottae. protocols.io https://dx.doi.org/10.17504/protocols.io.ibucanw | 2021-04-15 09:15:50 | |||
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How to make your protocol more reproducible, discoverable, and user-friendly Resource Report Resource Website |
protocols.io team | DOI:10.17504/protocols.io.g7vbzn6 | VERVE Net, protocols.io news, Protist Research to Optimize Tools in Genetics (PROT-G) | Protocols.io offers extensive flexibility in entering and presenting detailed methods. However, simple suggestions in here can help to make your protocol more useful to you and others. Importantly, easy steps outlined here can greatly increase the visibility of your protocol and ensure that scientists who need can find it.If you have any questions, please feel free to reach out to the protocols.io team by e-mailing us at info [at] protocols.io. | protocols.io | 1 | 2019 | protocols.io team 2019. How to make your protocol more reproducible, discoverable, and user-friendly. protocols.io https://dx.doi.org/10.17504/protocols.io.g7vbzn6 | 2021-04-15 09:15:55 | |||
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Euplotes crassus micronuclear enrichment by PFGE Resource Report Resource Website |
Rachele Cesaroni, Angela Piersanti | DOI:10.17504/protocols.io.zk7f4zn | Protist Research to Optimize Tools in Genetics (PROT-G) | Universität Bern, University of Camerino | 1 | 2019 | Rachele Cesaroni, Angela Piersanti 2019. Euplotes crassus micronuclear enrichment by PFGE. protocols.io https://dx.doi.org/10.17504/protocols.io.zk7f4zn | 2021-04-15 09:16:00 |
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