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Authors: Colin Daniel

Proper citation: Colin Daniel 2020. Ab Lysis Buffer . protocols.io https://dx.doi.org/10.17504/protocols.io.bft7jnrn Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.

Proper citation: Sam Li 2020. MojoSort™ Mouse CD8a Selection Kit Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bbugintw Copy   

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Authors: Maria Streltsova, Sofya Erokhina, Leonid Kanevskiy, Dean Lee, William Telford, Elena Kovalenko
Summary: Here we present the simple and effective method for obtaining clones of NK cells, sorted according to a given set of surface markers. The lifespan of clones grown by this method can reach 14 weeks and cloning efficiency is up to 50%, depending on the original NK cell subset. Obtained clones are functionally active; they are able to lyse target cells and produce IFNγ. The principle of method includes single cell sorting and further clonal expansion in the presence of IL-2 and gene-modified K562 feeder cells. Our method allows to expand the progeny of a single NK cell up to 10-20×106 NK cells suitable to study phenotype, proliferative and functional activity of a certain NK cell clone.The work was supported by Russian Science Foundation, grant #16-15-00309.

Proper citation: Maria Streltsova, Sofya Erokhina, Leonid Kanevskiy, Dean Lee, William Telford, Elena Kovalenko 2018. Obtaining of NK cell clones using IL-2 and gene-modified K562 cells expressing membrane-bound IL-21. protocols.io https://dx.doi.org/10.17504/protocols.io.v87e9zn Copy   

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Authors: Katy Monteith
Group: Vale Lab
Summary: This protocol describes how to systemically infect fruit flies with either a bacterial or viral infection through pricking the mesopleuron of the thorax

Proper citation: Katy Monteith 2018. systemic infection in Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.mfrc3m6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2014. M0368 RT Mixture. protocols.io https://dx.doi.org/10.17504/protocols.io.chwt7d Copy   

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Authors: Bonnie Hurwitz and Murat Meren
Group: Hurwitz Lab, MetaFunc Course
Summary: This protocol describes basic functions to view your contigs and profiles databases in Anvi'o.

Proper citation: Bonnie Hurwitz and Murat Meren 2016. MG_HW12: Anvi'o interactive. protocols.io https://dx.doi.org/10.17504/protocols.io.gg8btzw Copy   

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Authors: Elis Sales Muniz Lima, Ana Cesarina Silva Gomes, Hyara Brena Oliveira Rufino, Jamile Xavier de Oliveira

Proper citation: Elis Sales Muniz Lima, Ana Cesarina Silva Gomes, Hyara Brena Oliveira Rufino, Jamile Xavier de Oliveira 2020. Mulheres na praça, saúde de graça: relato de experiência da equipe multiprofissional do grupo de mulheres do bairro Tamarindo. protocols.io https://dx.doi.org/10.17504/protocols.io.bay4ifyw Copy   

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Authors: Michal Ronikier, Tomasz Suchan
Group: Molecular Biogeography Group

Proper citation: Michal Ronikier, Tomasz Suchan 2020. AFLP RedTaq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rpvd5n6 Copy   

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Authors: Ian Mackay
Summary: This version is based on a recipe (see forked version) my team and I used this assay between 2008-2015; we dubbed it the 'Nix assay'. It targets the 5'UTR and employs quite degenerate oligos.In silico sequence alignments indicated the olignucleotides could theoretically detect aft least HPeV 1-7, 17 and 18. Further validation of this recipe is ongoing.However during a period of assay comparison, another assay (see link below), the 'Benschop assay' (J.Clin.Virol. 2008. 41(2):69-74), was found to produce more sigmoidal and higher curves and 1-5 cycle improvements to CT values when compared among the sample sampe extract set.

Proper citation: Ian Mackay 2017. Human Parechovirus A real-time RT-PCR ["Nix assay"; 2017-]. protocols.io https://dx.doi.org/10.17504/protocols.io.k2kcycw Copy   

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Authors: Frank Ferrer-Gonzalez, Christa Smith
Summary: This protocol is a modified version of Bryn Durham et al., 2015 for extracting RNA from filters. This protocol has been tested with 0.22 μm pore size Durapore filters and 2.0 μm polycarbonate filters. Custom synthesized RNA transcript standards are added at the time of extraction and are recovered post-sequencing for quantitative metatranscriptome analysis (Satinsky et al., 2013). To prevent RNA degradation, work in a clean environment with asceptic practices and avoid possible introduction of RNases to samples and reagents.

Proper citation: Frank Ferrer-Gonzalez, Christa Smith 2020. RNA extraction and precipitation for 47 mm PES and polycarbonate filters. protocols.io https://dx.doi.org/10.17504/protocols.io.bervjd66 Copy   

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Authors: Chang-Ming Bai
Summary: This protocol is used to outline the process of Nanopore library preparation for Scapharca broughtonii genome.

Proper citation: Chang-Ming Bai 2019. The Nanopore libraries preparation for the Scapharca broughtonii. protocols.io https://dx.doi.org/10.17504/protocols.io.z8bf9sn Copy   

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Authors: Hayley Templar, Sandra McLellan
Summary: A-2. ISCO Sampling ProtocolRiver and estuary samples will be collected using an automated ISCO sampler using the following protocol:Sampler Description:A Teledyne ISCO 3700 full size portable sequential sampler is used for sampling.ISCO sampler consists of a rosette of 24, one-liter polypropylene bottles in which samples are collected at regular intervals.Includes a pump, which pumps water through a ¼-inch, flexible sample line into sample bottles. Sample line is automatically rinsed before each new sample is collected.Controller on ISCO sampler allows for sample programming. Sampler Installation:The ISCO samplers are installed within a sturdy, locked enclosure that is protected from damage due to tampering and environmental hazards (usually installed within a U.S. Geological Survey gaging station).ISCO samplers are installed by extending the ¼-inch suction line from the sampler into the water body being sampled.Sample line is secured using PVC pipe attached to a permanent structure, such as a river wall or concrete river bed.A Campbell® Scientific CR1000 datalogger and AirLink® LS300 Raven cell modem is installed to communicate with the ISCO, allowing for remote control and monitoring of the sampler.ISCO samplers are generally programmed to collect 250 mL every 15 minutes for up to 24 hours (each bottle represents a composite, one-hour sample). Field Collection:ISCO samplers are turned on remotely using the Campbell® Scientific LoggerLink mobile application, which communicates with the datalogger.Sampler is filled with ice to ensure sample preservation and ice is replaced once per day while the sampler is running. When samples are collected, if ice is melted, the temperature of the melted ice water is recorded on a field sheet. Water samples are collected in two-liter plastic Nalgene bottles, which are sanitized with dish soap after each use. A small quantity of sample water from each sample bottle is poured into a Nalgene bottle, capped, shaken, and dumped to remove any soap residue from the bottle before the sample is collected (x3). During and within 24 hours of an event, two ISCO sample bottles are combined into one, two-liter Nalgene bottle when the samples are collected (results in a two hour composite sample). 24 hours after an event, four ISCO sample bottles are combined into one, two-liter Nalgene bottle when the samples are collected (results in a four hour composite sample). Samples are immediately placed on ice and taken back to the lab for filtering. One-liter ISCO sample bottles are rinsed with deionized water (x3) and replaced in the sampler after each sample is removed.Bottle blanks are collected once per event at each site by collecting a sample of deionized water using the same procedure that a field sample would be collected.Sampler Installation:The ISCO samplers are installed within a sturdy, locked enclosure that is protected from damage due to tampering and environmental hazards (usually installed within a U.S. Geological Survey gaging station).ISCO samplers are installed by extending the ¼-inch suction line from the sampler into the water body being sampled.Sample line is secured using PVC pipe attached to a permanent structure, such as a river wall or concrete river bed.A Campbell® Scientific CR1000 datalogger and AirLink® LS300 Raven cell modem is installed to communicate with the ISCO, allowing for remote control and monitoring of the sampler.ISCO samplers are generally programmed to collect 250 mL every 15 minutes for up to 24 hours (each bottle represents a composite, one-hour sample). Field Collection:ISCO samplers are turned on remotely using the Campbell® Scientific LoggerLink mobile application, which communicates with the datalogger.Sampler is filled with ice to ensure sample preservation and ice is replaced once per day while the sampler is running. When samples are collected, if ice is melted, the temperature of the melted ice water is recorded on a field sheet. Water samples are collected in two-liter plastic Nalgene bottles, which are sanitized with dish soap after each use. A small quantity of sample water from each sample bottle is poured into a Nalgene bottle, capped, shaken, and dumped to remove any soap residue from the bottle before the sample is collected (x3). During and within 24 hours of an event, two ISCO sample bottles are combined into one, two-liter Nalgene bottle when the samples are collected (results in a two hour composite sample). 24 hours after an event, four ISCO sample bottles are combined into one, two-liter Nalgene bottle when the samples are collected (results in a four hour composite sample). Samples are immediately placed on ice and taken back to the lab for filtering. One-liter ISCO sample bottles are rinsed with deionized water (x3) and replaced in the sampler after each sample is removed.Bottle blanks are collected once per event at each site by collecting a sample of deionized water using the same procedure that a field sample would be collected.Field Collection:ISCO samplers are turned on remotely using the Campbell® Scientific LoggerLink mobile application, which communicates with the datalogger.Sampler is filled with ice to ensure sample preservation and ice is replaced once per day while the sampler is running. When samples are collected, if ice is melted, the temperature of the melted ice water is recorded on a field sheet. Water samples are collected in two-liter plastic Nalgene bottles, which are sanitized with dish soap after each use. A small quantity of sample water from each sample bottle is poured into a Nalgene bottle, capped, shaken, and dumped to remove any soap residue from the bottle before the sample is collected (x3). During and within 24 hours of an event, two ISCO sample bottles are combined into one, two-liter Nalgene bottle when the samples are collected (results in a two hour composite sample). 24 hours after an event, four ISCO sample bottles are combined into one, two-liter Nalgene bottle when the samples are collected (results in a four hour composite sample). Samples are immediately placed on ice and taken back to the lab for filtering. One-liter ISCO sample bottles are rinsed with deionized water (x3) and replaced in the sampler after each sample is removed.Bottle blanks are collected once per event at each site by collecting a sample of deionized water using the same procedure that a field sample would be collected.

Proper citation: Hayley Templar, Sandra McLellan 2018. ISCO Collection - AOC Project. protocols.io https://dx.doi.org/10.17504/protocols.io.prrdm56 Copy   

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Authors: Saurabh Mehta, Bryan Gannon, Camille E. Jones, Laura S. Hackl, Shoba A. Udipi, Varsha Thakker, Aparna Thorat, Wesley Bonam, Jere D. Haas, Julia Finkelstein
Group: Mehta Research Group @ Cornell

Proper citation: Saurabh Mehta, Bryan Gannon, Camille E. Jones, Laura S. Hackl, Shoba A. Udipi, Varsha Thakker, Aparna Thorat, Wesley Bonam, Jere D. Haas, Julia Finkelstein 2020. Effect of Multiple Biofortified Food Crops-based Meals on Micronutrient Status and Immune Function in The First 1000 Days of Life in India: Study Protocol for a Randomized Controlled Feeding Trial. protocols.io https://dx.doi.org/10.17504/protocols.io.bqcmmsu6 Copy   

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Authors: Deepak Nanjappa and Kathryn Coyne
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 2019. Glass bead transformation of Heterosigma akashiwo. protocols.io https://dx.doi.org/10.17504/protocols.io.4qhgvt6 Copy   

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Authors: Vinay Saunders, David I Wilson
Summary: Procotol describing RNA extraction from dissected human tissue followed by reverse transcription to cDNA, PCR for detection of TMSB4X and separation of PCR products by agarose gel.

Proper citation: Vinay Saunders, David I Wilson 2018. RNA extraction, reverse transcription and PCR for TMSB4X. protocols.io https://dx.doi.org/10.17504/protocols.io.rvbd62n Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: The adiponectin enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay for the detection of total adiponectin levels in serum, adipocyte extracts, and cell culture media samples. This experiment uses a 96-well plate coated with anti-adiponectin antibodies and developed with a secondary anti-adiponectin antibody linked to horseradish peroxidase. The ELISA is developed with 3,3',5,5'-Tetramethylbenzidine (TMB), a chromogenic substrate and read on an automated plate spectrophotometer at 450 nm. Adiponectin is an adipocyte-derived hormone (adipokine) that regulates insulin sensitivity and glucose metabolism.

Proper citation: Jason Kim 2019. U Mass - Adiponectin. protocols.io https://dx.doi.org/10.17504/protocols.io.xm9fk96 Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for generating stable cell lines with lentivirus. To see the full abstract and additional resources, please visit the Addgene protocol page.Sample Data

Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Generating Stable Cell Lines with Lentivirus. protocols.io https://dx.doi.org/10.17504/protocols.io.4xtgxnn Copy   

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Authors: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom
Group: Coronavirus Method Development Community
Summary: SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Proper citation: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom 2020. Pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays. protocols.io https://dx.doi.org/10.17504/protocols.io.bfghjjt6 Copy   

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Authors: Elizabeth Neumann, Heath Patterson, Jamie Allen, Maya Brewer, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:Obtain autofluorescence microscopy images of tissues.Expected Outcome:An RGB autofluorescence microscopy image of the tissue section that enables registration and correlation of different imaging modalities on a pixel by pixel basis.

Proper citation: Elizabeth Neumann, Heath Patterson, Jamie Allen, Maya Brewer, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2019. Autofluorescence Microscopy Data Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.7e3hjgn Copy   

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Authors: Ruth Timme, Sai Laxmi Gubbala Venkata, Maria Balkey, Robyn Randolph, William Wolfgang, Errol Strain
Group: GenomeTrakr
Summary: PURPOSE: Step-by-step instructions for checking WGS sequence quality. The MicroRunQC workflow, implimented in a custom Galaxy instance, will produce quality assessments for raw reads (illumina paired-end fastq files) and draft de novo assemblies, along with reporting the sequence type for each isoalte. This workflow will work on most microbial pathogens, so we advise laboratories to upload their entire MiSeq/NextSeq run through this workflow. SCOPE: This protocol covers the following tasks:1. set up an account in GalaxyTrakr2. Create a new history/workspace3. Upload data4. Execute the MicroRunQC workflow5. Interpret the results

Proper citation: Ruth Timme, Sai Laxmi Gubbala Venkata, Maria Balkey, Robyn Randolph, William Wolfgang, Errol Strain 2020. Assessing sequence quality in GalaxyTrakr. protocols.io https://dx.doi.org/10.17504/protocols.io.bdvfi63n Copy   

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