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Authors: Ben Humphreys, Yuhei Kirita
Group: Human Cell Atlas Method Development Community
Summary: This protocol is based upon Habib, N et al., Nature Methods 14: 955-958, 2017  and Basu et al., Protocol Exchange 2017: DroNc-seq step-by-step with adaptions for adult human kidney including: Tissue mincing, homogenization strokes, addition of protease inhibitor and RNasin and adjustments to strainer size and sequence.

Proper citation: Ben Humphreys, Yuhei Kirita 2018. Nuclei isolation from human kidney for single-nucleus RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.nahdab6 Copy   

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Authors: Tamara Matute, Isaac Núñez, Fernan Federici
Group: Laboratorio de Tecnologias Libres, Reclone.org (The Reagent Collaboration Network)
Summary: This protocol collection specifies how to carry out LAMP or RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) reactions with homemade reagents and home-brewed enzymes.It describes how to prepare all the solutions and buffers to perform the reactions.This collection was developed in the context of COVID-19 pandemic to provide access to RT-LAMP tools in accordance with the principles of ReClone community initiative. It is the result of information exchange and reagent sharing with a huge community of collaborators from ReClone, gLAMP and JOGL.

Proper citation: Tamara Matute, Isaac Núñez, Fernan Federici 2021. Low Cost LAMP and RT-LAMP. protocols.io https://dx.doi.org/10.17504/protocols.io.bsejnbcn Copy   

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Authors: Kendra Negrey
Summary: Describes collection for the following:Domoic acid (particulate and dissolved)*ChlorophyllDNANutrients: NH4, urea, N, P, SiFlow cytometer counts (Synecocccus, pico-eukaryotes, heterotrophic bacteria)Whole water/net tow archiveWhole cell probe counts (P. australis, P. multiseries, Alexandrium)*Filters are also used for HPLC pigment analysis

Proper citation: Kendra Negrey 2018. SC Wharf - water processing (in lab). protocols.io https://dx.doi.org/10.17504/protocols.io.rxkd7kw Copy   

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Authors: Muofhe Tshibalanganda, Anton Du Plessis, Stephan Le Roux
Summary: This protocol outlines the steps followed to determine a potential "killer pore" in the area of failure of DMLS Ti6Al4V specimen. The methodology can be used more generally for positively identifying and quantifying small isolated pores in additively manufactured metals. In this case the focus was on analysis of a 4 mm region near the eventual fracture surface, to identify all pores in the vicinity of the "killer pore" - the one causing failure by fatigue tests. This protocol was developed for Ti6Al4V with 99.99% density (only small amount of porosity, very small pores only), tensile fatigue specimen round dogbone geometry with 5 mm gauge width.

Proper citation: Muofhe Tshibalanganda, Anton Du Plessis, Stephan Le Roux 2019. Serial killer pore investigation: protocol for analyzing fatigue failure pores in additively manufactured Ti6Al4V. protocols.io https://dx.doi.org/10.17504/protocols.io.yebftan Copy   

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Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
Summary: SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets.Version 2 includes: Optimized thermocycler conditionsLocally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclersUse of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates.Version 3 has been updated to remove steps for sample self-collection.Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers.

Proper citation: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie 2020. SalivaDirect™: RNA extraction-free SARS-CoV-2 diagnostics. protocols.io https://dx.doi.org/10.17504/protocols.io.bkjgkujw Copy   

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Authors: Charlotte Scott, Martin Guilliams
Group: Human Cell Atlas Method Development Community
Summary: Optimised protocol for the isolation of non-parenchymal cells from human liver biopsies. Used on 1-2cm3 biopsies.

Proper citation: Charlotte Scott, Martin Guilliams 2018. Protocol for the isolation of non-parenchymal liver cells from human liver biopsies (1-2cm3). protocols.io https://dx.doi.org/10.17504/protocols.io.viwe4fe Copy   

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Authors: Alexander Chamessian
Summary: This protocol details the efficient isolation of intact nuclei from frozen peripheral nerve from human (e.g. sciatic, tibial, etc.)

Proper citation: Alexander Chamessian 2019. Isolation of Nuclei from Frozen Human Peripheral Nerve. protocols.io https://dx.doi.org/10.17504/protocols.io.6zqhf5w Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes how to make Artificial Cerebrospinal Fluid VIII (ACSF.VIII). ACSF.VIII is used for multiple applications including incubation of fresh human brain slices prior to electrophysiological recording.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. Artificial Cerebrospinal Fluid VIII (ACSF.VIII). protocols.io https://dx.doi.org/10.17504/protocols.io.bdpai5ie Copy   

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Authors: Elena Hilario
Group: Plantae
Summary: Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The details about how each approach performs can be found in the citation shown below.This protocol explains how to prepare each type of GBS library, with comments on expected results and controls needed to confirm each step worked.

Proper citation: Elena Hilario 2017. Genotyping by Sequencing (GBS) library protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.kzmcx46 Copy   

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Authors: Brittany Zepernick, Matthew Saxton, Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Great Lakes Center for Fresh Waters and Human Health
Summary: This method can be used to assess and quantify the rate of silica deposition (bSi) over time in diatoms to determine their rate of frustule synthesis. This protocol has been adapted for the processing of both cultures as well as environmental samples when inoculated with PDMPO [2‐(4‐pyridyl)‐5‐((4‐(2dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole], which is a fluorescent dye that is co-deposited with silica during frustule biosynthesis in a 3230:1 Si:PDMPO (mol:mol) ratio.

Proper citation: Brittany Zepernick, Matthew Saxton, Steven Wilhelm 2019. Quantifying Biogenic Silica (bSi) Deposition Rates Adapted Method & Fluorescence Reading (PDMPO) via Fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.735hqq6 Copy   

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Authors: Angel Justiz-Vaillant, Belkis Ferrer-Cosme
Group: University of the West Indies, [email protected]
Summary: MCP-1 is believed to play an important role in monocyte infiltration into tumor tissues. [1] MCP-1 plays an important role in the pathogenesis of atherosclerosis. There is considerable evidence that supports that monocytes containing MCPs and macrophages influence the growth of other cell types within the atherosclerotic lesion. [2]References1. Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29(6):313-326. doi:10.1089/jir.2008.00272. Lin J, Kakkar V, Lu X. Impact of MCP-1 in atherosclerosis. Curr Pharm Des. 2014;20(28):4580-4588. doi:10.2174/1381612820666140522115801

Proper citation: Angel Justiz-Vaillant, Belkis Ferrer-Cosme 2020. ELISA for measurement of monocyte chemoattractant protein-1 (MCP-1/CCL2) in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj7fkrjn Copy   

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Authors: David A. Antonetti
Group: Mouse Metabolic Phenotyping Centers
Summary: A virtual optometry system is used to quantify the spatial vision of laboratory animal.

Proper citation: David A. Antonetti 2019. U Michigan - Optokinetic Measurements of Visual Acuity and Contrast Sensitivity. protocols.io https://dx.doi.org/10.17504/protocols.io.x99fr96 Copy   

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Authors: Fabian Fink, 26.08.2014 modified after Edwars et al., 1991 Nucleic Acids Research

Proper citation: Fabian Fink, 26.08.2014 modified after Edwars et al., 1991 Nucleic Acids Research 2016. Extraction of genomic DNA using "Edwards" buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.grdbv26 Copy   

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Authors: Daniel Groelz, Nadine Dettmann
Summary: Quantitative PCR for amplification of genomic DNA from FFPE (formalin-fixed and paraffin-embedded), PFPE (PAXgene Tissue-fixed and paraffin-embedded), and snap-frozen fixed tissues.

Proper citation: Daniel Groelz, Nadine Dettmann 2018. Rat ACTB one-step qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.qnidvce Copy   

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Authors: Steven Wilhelm, Samantha Coy, Alyssa Alsante
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Adapted from: Ortmann and Suttle (2009) Determination of Virus Abundance by Epifluorescence microscopy. Ch. 10 Methods of Molecular Biology. Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol.

Proper citation: Steven Wilhelm, Samantha Coy, Alyssa Alsante 2018. Epifluorescent Microscopy of Virus Particles Using SYBR Green. protocols.io https://dx.doi.org/10.17504/protocols.io.qsgdwbw Copy   

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Authors: William H. Wilson and Declan Schroeder
Group: VERVE Net

Proper citation: William H. Wilson and Declan Schroeder 2016. Generation of DNA fragments by sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.dy57y5 Copy   

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Authors: Momoko Ogitani

Proper citation: Momoko Ogitani 2017. immunofluorescence analysis of myosin heavy chain isoforms. protocols.io https://dx.doi.org/10.17504/protocols.io.iwjcfcn Copy   

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Authors: Remco Stam

Proper citation: Remco Stam 2016. Cultivation / Spore production for Fusarium spp. protocols.io https://dx.doi.org/10.17504/protocols.io.fmtbk6n Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: This IgY purification technology can be used to purified IgY from the egg yolk of any bird as ducks, bantam hens, ostrich, chicken, pigeon, pheasant, quail, guinea hen, cattle egret and goose.

Proper citation: Angel Justiz-Vaillant 2020. Purification of the any avian IgY using chloroform, trichloroacetic acid and affinity purification using HiTrap™ Columns.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjnskmee Copy   

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Authors: Olin Silander
Summary: For CRISPR-cas9 protocols including BAC-PULCE, T7 polymerase is used to transcribe the crRNA and tracrRNA to make dgRNA for cas9. The two components of the dual guides are the crRNA (containing your variable 20 nt target plus a 22 nt constant region) and the tracrRNA (a 72 nt constant region). We used a combination of these protocols: https://www.protocols.io/view/in-vitro-transcription-for-dgrna-3bpgimn /dx.doi.org/10.17504/protocols.io.3bpgimnhttps://international.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050Nanopore protocol Cas-mediated PCR-free enrichment - please refer to this protocol in particular for further important detail around sequencing the DNA library. Using modified “In Vitro Transcription for dgRNA V.2” (Lyden et al 2019):In this protocol, we are designing crRNAs against which Cas9 will target. Using this sequence, we will add a T7 RNA polymerase binding site at the 5’ end, a 3’ tracrRNA binding site, and use the reverse complement of this to order our DNA oligos (below more details):Designing crRNAs using CHOPCHOPv3 (Labun et al 2019), insert your gene target and CHOPCHOP will find a sequence of 20nt’s that will end in an  ‘NGG-3’5’----NNNNNNNNNNNNNNNNNNNNNGG----3’where 20Ns are your target site. Cas9 cuts between the 17th and 18th N of your target.The sequence of each crRNA should be as follows, with the Ns replaced by your 20 nt target:TAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGThe underline portion is the T7 RNA polymerase binding site. The addition at the 3’ end represents the region to which an 18mer will bind, in order to allow T7 a double stranded binding site.18mer T7:TAATACGACTCACTATAG

Proper citation: Olin Silander 2021. In vitro transcription of crRNA and tracrRNA from DNA oligos for cas9 enrichment and nanopore sequencing (for Bac - PULCE)  . protocols.io https://dx.doi.org/10.17504/protocols.io.brbnm2me Copy   

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