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Authors: Adriana Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker
Group: Tara Oceans
Summary: The protocols in this collection are from the Alberti A., et al manuscript (Alberti A. 2017, Scientific Data). These protocols provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). This collection complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world’s planktonic ecosystems.'From the Methods section:'The generation of information-rich data from marine plankton samples presents unique challenges that are inherent to the particular sampling conditions at sea and the wide spectrum of organisms included in that environment. All processing steps, including biomass collection, sample preservation, nucleic acids extractions and sequencing library preparation, are critical and require specific protocols and robust methods in order to ensure comparability of results and limit potential biases.Our methods were either developed specifically for Tara Oceans samples or carefully selected among existing ones in order to meet the requirements of our sequencing strategy and to produce optimized datasets for downstream bioinformatics analyses, as for example the production of overlapping reads from metagenomics libraries to facilitate assembly. They are presented in five sub-sections, starting with a brief description of how samples were handled between the research vessel and the processing laboratories (protocol 1). Protocol 2 reports on DNA and RNA extractions procedures for -omics analyses, including the generation of amplified genomic DNA from uncultured isolated unicellular eukaryotes. The generation of 18S and 16S rRNA amplicons from DNA of specific size-fractions is described in protocol 3 and Illumina libraries preparation in protocol 4. Sequencing procedures and post-sequencing data processing are described in protocol 5. For details on the onboard sampling protocols, see Pesant et al.'

Proper citation: Adriana Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker 2020. Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. protocols.io https://dx.doi.org/10.17504/protocols.io.qv6dw9e Copy   

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Authors: Jacopo Niccolo Cerasoni
Summary: Lithic illustrations are often used in scientific publications to efficiently communicate the technological and morphological characteristics of stone tools. They offer invaluable information and insights not only on how stone raw materials were transformed into their final form, but also on the individuals that made them. Here, the “Stone Tools Illustrations with Vector Art” (STIVA) Method is presented, which involves the illustration of lithic artefacts using vectorial graphics software (Adobe® Illustrator®). This protocol follows an optimised step-by-step method, presenting ten major sections that constitute the creation of a lithic illustration: photography, vectorial software configuration, scale, outline, scar borders, ripples, cortex, symbols, composition, and export. This method has been developed to allow researchers, students and educators to create clear and competent illustrations for any application, from scientific publications to public outreach.

Proper citation: Jacopo Niccolo Cerasoni 2020. Stone Tools Illustrations with Vector Art: The 'STIVA' Method. protocols.io https://dx.doi.org/10.17504/protocols.io.bnbtmann Copy   

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Authors: Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: A protocol for recovering viable single cell suspensions from cryopreserved human nasopharyngeal swabs for downstream applications, such as single-cell RNA-seq. The illustrated schematic below details the process.

Proper citation: Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes 2020. Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhkkj4w Copy   

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Authors: Angel Justiz-Vaillant
Group: Carbon
Summary: This ELISA was used to study the interactions between Staphylococcal protein-A (SpA) and protein-LAG (PLAG) with different immunoglobulin preparations from mammalian and avian species.

Proper citation: Angel Justiz-Vaillant 2021. Staphylococcal Protein-A and Chimeric Protein-LAG sandwich ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.bta5nig6 Copy   

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Authors: Chise Suzuki, Reiji Aoki, Ayako Aoki-Yoshida
Summary: The immunostaining detection of isolated lymphoid follicles (ILFs) was performed according to the method of McDonald and Newberry [BioTechniques. 2007; 43(1): 50-56], with some modifications. Instead of mounting the tissue samples on a mounting plate, each segment was treated in a 15 ml tube in each step. DAB stained intestines were finally mounted on a glass slide using an aqueous mounting medium.

Proper citation: Chise Suzuki, Reiji Aoki, Ayako Aoki-Yoshida 2017. Immunostaining of the isolated lymphoid follicles (ILFs) in the whole small intestine. protocols.io https://dx.doi.org/10.17504/protocols.io.jr3cm8n Copy   

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Authors: tenOever Lab
Group: Coronavirus Method Development Community
Summary: Method to plaque the SARS-CoV-2 virus Image attribution:Y tambe / CC BY-SA (http://creativecommons.org/licenses/by-sa/3.0/)Downloaded from: https://commons.wikimedia.org/wiki/File:Plaque_assay_macro.jpg

Proper citation: tenOever Lab 2020. SARS-CoV-2 virus plaque assays [Biosafety Level 3]. protocols.io https://dx.doi.org/10.17504/protocols.io.bdtni6me Copy   

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Authors: Adam Vivian-Smith, Jørn Henrik Sønstebø
Summary: Double digested Restriction-site Associated DNA (ddRAD) sequencing is a powerful approach for identifying and analyzing genome-wide SNP variation. Many studies have now used ddRAD protocols for population genetic studies. Here we have adapted the protocol from Peterson (2012) for Ion Torrent sequencing to produce a significantly streamlined workflow capable of having fully sequenced ddRAD libraries in two days. A reduced number of steps for producing a ddRAD library is achieved through the use of a unidirectional double digestion-ligation reaction with adaptors having fusion barcodes in the 5' and 3' ends. This also allows for the immediate pooling of sets of compatible barcoded samples for downstream processing. The A-adaptor contains the standard Ion Torrent barcodes and a key sequence, while the P1-adaptor contains a divergent Y-adaptor with a paired-end code for increased multiplexing. The described system and adaptors are compatible with the SbfI, PstI and NsiI restriction endonucleases in the A adaptor, while the P1-adaptor has compatible overhangs with NdeI or MseI, and, MspI or HpaII. The later two restriction enzymes were chosen as isoschizomers for their differentially sensitivity to CpG methylation, thus allowing the use of this protocol for epigenetic ddRAD profiling at genomic loci with 5-methylcytosine and 5-hydroxymethylcytosine modifications. This protocol takes an advantage in that the Ion Torrent platform has scalability with different sequencing chip sizes, and that the protocol has a range of compatible restriction endonucleases with different motif lengths. This ensures a versatile, cost-effective and flexible method to which you can tune the number of ddRAD loci being profiled, for both small and large genomes, with relative speed and with the ability to observe the performance from small pilot scale reactions.

Proper citation: Adam Vivian-Smith, Jørn Henrik Sønstebø 2017. A streamlined ddRAD tag protocol for use with the Ion Torrent sequencer, as a versatile probe for populations, genetics and genomics. protocols.io https://dx.doi.org/10.17504/protocols.io.khuct6w Copy   

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Authors: Elizabeth Fozo

Proper citation: Elizabeth Fozo 2020. 1.2 Bradford assay. protocols.io https:// Copy   

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Authors: Kamron Mojabe
Group: UCSC BME 22L

Proper citation: Kamron Mojabe 2020. Copy of Lab 1 Notebook. protocols.io https:// Copy   

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Authors: Vladimir Vigdorovich
Summary: This is a protocol for plasmid minipreps that does not involve any columns and uses commonly accessible reagents. While it doesn't result in highly pure DNA, it can provide a sufficient quantity of plasmid for sequencing, restriction analysis, preparative digests.Beware of the large amount of RNA that co-purifies with plasmid DNA (it acts as an efficient carrier during the nucleic acid precipitation step). This contamination will make it impossible to determine DNA concentration from standard 280-nm absorbance readings. Credits: I first learned this protocol from the old-style protocols for bacmid purification using the Bak-to-Bac system (Invitrogen). In that system, the large (>50 kb) bacmid molecules were deemed too fragile for the Qiagen spin columns.

Proper citation: Vladimir Vigdorovich 2016. Column-free plasmid miniprep. protocols.io https://dx.doi.org/10.17504/protocols.io.d7e9jd Copy   

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Authors: Jennifer Brum
Group: VERVE Net, Sullivan Lab
Summary: Purpose: This protocol describes how to analyze a natural aquatic virus sample using transmission electron microscopy (TEM). The purpose is to obtain the capsid diameter distributions of the viral assemblage, tail length distributions of the viral assemblage, and percentages of each viral morphotype in a sample.Note: Prior to using this protocol, deposit viruses from your sample onto a TEM grid using the protocol “Quantitatively Depositing Viruses onto TEM Grids” then stain the viruses using the protocol “Positive and Negative Staining of Viruses on TEM Grids”.

Proper citation: Jennifer Brum 2016. Analysis of Viral Morphological Characteristics. protocols.io https://dx.doi.org/10.17504/protocols.io.dde23d Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for inoculating a liquid bacterial culture. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/inoculate-bacterial-culture/.

Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Inoculating a Liquid Bacterial Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.5abg2an Copy   

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Authors: Anna Behle, Miriam D, Susanne Vollmer
Group: iGEM Duesseldorf 2018
Summary: This protocol describes how to prepare a 100x BG-11 +N stock.1x BG-11 media could be created from the stock and supplemental stocks and trace metals could be added afterwards.This protocol is based on Anne Behle M.Sc. Reciepe for standard BG-11 media protocol.Recipes for standard and alternative BG11 for culturing freshwater cyanobacteria, such as Synechocystis sp. PCC 6803, as described.Media is usually not suitable for marine cyanobacteria.

Proper citation: Anna Behle, Miriam D, Susanne Vollmer 2018. 100x BG-11 media. protocols.io https://dx.doi.org/10.17504/protocols.io.n97dh9n Copy   

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Authors: Gonzalo Vidal-Peña, Tim Rudge
Summary: Corchea is a paper-based microfluidic system for culture and comunicate bacteria on flow. Its has a hardware that you can make with a 3D printer and laser cut. The system allows the directed comunications between biofilms and we aim to array biological logic gates on it to make complex information processing.

Proper citation: Gonzalo Vidal-Peña, Tim Rudge 2018. Corchea: paper-based microfluidic device. protocols.io https://dx.doi.org/10.17504/protocols.io.vtwe6pe Copy   

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Authors: Neilier Junior
Summary: DNA and RNA stabilization solution is an aqueous and non-toxic tissue storage reagent that rapidly permeates tissues to stabilize and protect nucleic acids in fresh specimens. Tissue pieces are harvested and immediately submerged in stabilization solution for storage. It can be used for DNA and RNA preservation with most tissues, cultured cells and is compatible with most DNA/RNA isolation methods. Proteins are precipitated, but can still be used for analyzes that do not require their native structure, such as western blotting or gel electrophoresis. Samples in DNA and RNA stabilization solution can be stored indefinitely in RNAlater Solution at –20 °C or below.

Proper citation: Neilier Junior 2020. DNA and RNA stabilization. protocols.io https://dx.doi.org/10.17504/protocols.io.be2pjgdn Copy   

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Authors: Magda Bienko, Nicola Crosetto, Leonid Teytelman, Sandy Klemm, Shalev Itzkovitz, and Alexander van Oudenaarden
Summary: This protocol describes the design of primer pairs against the human genome for the synthesis of probes for high-definition DNA FISH (HD-FISH). This pipeline selects PCR primer pairs with optimal thermodynamic features, delimiting amplicons 200–220 nucleotides in length, and filters out primer pairs amplifying multiple targets as well as cross-hybridizing amplicons. Using such primers, highly specific double-stranded probes can be rapidly generated for virtually any desired genomic locus by fluorescently labeling pooled amplicons after PCR.While this protocol describes the design against the human genome, we have also used it to geneate a genome-wide library for mouse. The design and method should work across other organisms as well.For more information please see the full paper (and the dedicated hdfish.eu website):Bienko, Magda et al. “A Versatile Genome-Scale PCR-Based Pipeline for High-Definition DNA FISH.” Nature methods 10.2 (2013): 122–124. PMC. Web. 9 Nov. 2015.

Proper citation: Magda Bienko, Nicola Crosetto, Leonid Teytelman, Sandy Klemm, Shalev Itzkovitz, and Alexander van Oudenaarden 2015. Design of genome-wide HD-FISH probes. protocols.io https://dx.doi.org/10.17504/protocols.io.d6m9c5 Copy   

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Authors: Lenny Teytelman, Jasper Rine
Summary: This is a modified protocol for URA3 counter-selection in S. cerevisiae and similar yeasts. It is identical to the standard counter-selection with two transformations, with the exception of the replica-plating step.This protocol is appropriate for genomic loci where URA3 is over-expressed and the Ura3 protein is likely to be present in the colonies and cells at higher toxic levels. Because this method required five times as many plates for the replica-plating, it is more wasteful and should only be used when necessary.

Proper citation: Lenny Teytelman, Jasper Rine 2020. Modified protocol for URA3 counter-selection at highly expressed regions of Saccharomyces cerevisiae. protocols.io https://dx.doi.org/10.17504/protocols.io.bf7njrme Copy   

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Authors: Fabien Vincent

Proper citation: Fabien Vincent 2017. cGAS Enzymatic Assay – cGAMP Mass Spectrometry Format. protocols.io https://dx.doi.org/10.17504/protocols.io.jazcif6 Copy   

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Authors: Maira Rubi, Maira Rubi Segura Campos
Summary: The assay was carried out following the protocol reported by Dineshkumar et al. [8]. Starch (2 mg) was suspended in a tube containing 0.2 mL of 0.5 M Tris-HCl (Sigma-Aldrich, USA) buffer (pH 6.9) with 0.01 M calcium chloride as substrate. The tube was boiled for 5 min and then preincubated at 37 °C for 5 min. Plant aqueous extract (1 mg) was dissolved with 1 mL of 0.1% of dimethyl sulfoxide in order to obtain a concentration of 1,000 µg/mL; then 0.2 mL of aqueous extract was added to the tube containing the substrate solution, 0.1 mL of porcine pancreatic amylase in Tris-HCl buffer (2 U/mL) was also added, and incubated for 10 min at 37 °C. Finally, the reaction was stopped with 0.5 mL of acetic acid (50% v/v) and centrifuged 5 min at 1,811 × g and 4 °C. The assay was performed in triplicate. The a-amylase inhibitory activity was calculated using the formula (Ac+) – (Ac-) – (As - Ab)/(Ac+) – (Ac-) × 100, where Ac+, Ac-, As, Ab are defined as the absorbance (595 nm) of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme), test sample (with enzyme), and a blank (a test sample without enzyme), respectively.

Proper citation: Maira Rubi, Maira Rubi Segura Campos 2018. In vitro α-amylase inhibitory assay. protocols.io https://dx.doi.org/10.17504/protocols.io.s9meh46 Copy   

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Authors: samz
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: samz 2020. AMDx Lateral Flow Assay WIth a Cover Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk54ky8w Copy   

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