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Nuclei isolation from human kidney for single-nucleus RNA-seq Resource Report Resource Website |
Ben Humphreys, Yuhei Kirita | DOI:10.17504/protocols.io.nahdab6 | Human Cell Atlas Method Development Community | 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. | Washington University in St. Louis, The Humphreys Lab | 1 | 2018 | 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 | 2021-04-15 09:15:46 | |||
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Low Cost LAMP and RT-LAMP Resource Report Resource Website |
Tamara Matute, Isaac Núñez, Fernan Federici | DOI:10.17504/protocols.io.bsejnbcn | Laboratorio de Tecnologias Libres, Reclone.org (The Reagent Collaboration Network) | 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. | Laboratorio de Tecnologías Libres, SynBioUC, SynBioUC | 1 | 2021 | Tamara Matute, Isaac Núñez, Fernan Federici 2021. Low Cost LAMP and RT-LAMP. protocols.io https://dx.doi.org/10.17504/protocols.io.bsejnbcn | 2021-04-15 09:15:46 | |||
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SC Wharf - water processing (in lab) Resource Report Resource Website |
Kendra Negrey | DOI:10.17504/protocols.io.rxkd7kw | 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 | University of California, Santa Cruz | 1 | 2018 | Kendra Negrey 2018. SC Wharf - water processing (in lab). protocols.io https://dx.doi.org/10.17504/protocols.io.rxkd7kw | 2021-04-15 09:15:19 | ||||
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Serial killer pore investigation: protocol for analyzing fatigue failure pores in additively manufactured Ti6Al4V Resource Report Resource Website |
Muofhe Tshibalanganda, Anton Du Plessis, Stephan Le Roux | DOI:10.17504/protocols.io.yebftan | 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. | CT scanner facility, CT scanner facility, CT scanner facility | 1 | 2019 | 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 | 2021-04-15 09:15:46 | ||||
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SalivaDirect™: RNA extraction-free SARS-CoV-2 diagnostics Resource Report Resource Website |
Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie | DOI:10.17504/protocols.io.bkjgkujw | Coronavirus Method Development Community | 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. | Vogels CB, Watkins AE, Harden CA, Brackney DE, Shafer J, Wang J, Caraballo C, Kalinich CC, Ott IM, Fauver JR, Kudo E, Lu P, Venkataraman A, Tokuyama M, Moore AJ, Muenker MC, Casanovas-Massana A, Fournier J, Bermejo S, Campbell M, Datta R, Nelson A, , Cruz CSD, Ko AI, Iwasaki A, Krumholz HM, Matheus J, Hui P, Liu C, Farhadian SF, Sikka R, Wyllie AL, Grubaugh ND, SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity. Med (New York, N.y.) doi: 10.1016/j.medj.2020.12.010 | Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Department of Environmental Sciences The Connecticut Agricultural Experiment Station; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Department of Epidemiology of Microbial Diseases, Yale School of Public Health | https://covidtrackerct.com/about-salivadirect/ | 5 | 2020 | 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 | 2021-04-15 09:15:19 | |
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Protocol for the isolation of non-parenchymal liver cells from human liver biopsies (1-2cm3) Resource Report Resource Website |
Charlotte Scott, Martin Guilliams | DOI:10.17504/protocols.io.viwe4fe | Human Cell Atlas Method Development Community | Optimised protocol for the isolation of non-parenchymal cells from human liver biopsies. Used on 1-2cm3 biopsies. | VIB-UGent Center for Inflammation Research, Ghent, Belgium, VIB-UGent Center for Inflammation Research, Ghent, Belgium | 1 | 2018 | 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 | 2021-04-15 09:15:20 | |||
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Isolation of Nuclei from Frozen Human Peripheral Nerve Resource Report Resource Website |
Alexander Chamessian | DOI:10.17504/protocols.io.6zqhf5w | This protocol details the efficient isolation of intact nuclei from frozen peripheral nerve from human (e.g. sciatic, tibial, etc.) | Washington University, Saint Louis | 1 | 2019 | Alexander Chamessian 2019. Isolation of Nuclei from Frozen Human Peripheral Nerve. protocols.io https://dx.doi.org/10.17504/protocols.io.6zqhf5w | 2021-04-15 09:15:20 | ||||
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Artificial Cerebrospinal Fluid VIII (ACSF.VIII) Resource Report Resource Website |
Allen Institute for Brain Science | DOI:10.17504/protocols.io.bdpai5ie | BICCN, Allen Institute for Brain Science | 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. | Allen Institute | 2 | 2020 | Allen Institute for Brain Science 2020. Artificial Cerebrospinal Fluid VIII (ACSF.VIII). protocols.io https://dx.doi.org/10.17504/protocols.io.bdpai5ie | 2021-04-15 09:15:20 | |||
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Genotyping by Sequencing (GBS) library protocols Resource Report Resource Website |
Elena Hilario | DOI:10.17504/protocols.io.kzmcx46 | Plantae | 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. | Kumar S, Hilario E, Deng CH, Molloy C, Turbocharging introgression breeding of perennial fruit crops: a case study on apple. Horticulture Research doi: 10.1038/s41438-020-0270-z | Plant and Food Research | http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143193 | 1 | 2017 | Elena Hilario 2017. Genotyping by Sequencing (GBS) library protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.kzmcx46 | 2021-04-15 09:15:47 | |
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Quantifying Biogenic Silica (bSi) Deposition Rates Adapted Method & Fluorescence Reading (PDMPO) via Fluorometer Resource Report Resource Website |
Brittany Zepernick, Matthew Saxton, Steven Wilhelm | DOI:10.17504/protocols.io.735hqq6 | The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Great Lakes Center for Fresh Waters and Human Health | 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. | University of Tennessee, Knoxville, Miami University of Ohio, The University of Tennessee, Knoxville | http://wilhelmlab.utk.edu/ | 1 | 2019 | 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 | 2021-04-15 09:15:20 | ||
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ELISA for measurement of monocyte chemoattractant protein-1 (MCP-1/CCL2) in human serum. Resource Report Resource Website |
Angel Justiz-Vaillant, Belkis Ferrer-Cosme | DOI:10.17504/protocols.io.bj7fkrjn | University of the West Indies, [email protected] | 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 | University of the West Indies St. Augustine, "Saturnino Lora Torres" Provincial Teaching Clinical Surgical Hospital. Cuba | 1 | 2020 | 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 | 2021-04-15 09:15:47 | |||
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U Michigan - Optokinetic Measurements of Visual Acuity and Contrast Sensitivity Resource Report Resource Website |
David A. Antonetti | DOI:10.17504/protocols.io.x99fr96 | Mouse Metabolic Phenotyping Centers | A virtual optometry system is used to quantify the spatial vision of laboratory animal. | University of Michigan - Ann Arbor | https://mmpc.org/shared/document.aspx?id=306&docType=Protocol | 1 | 2019 | 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 | 2021-04-15 09:15:21 | ||
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Extraction of genomic DNA using "Edwards" buffer Resource Report Resource Website |
Fabian Fink, 26.08.2014 modified after Edwars et al., 1991 Nucleic Acids Research | DOI:10.17504/protocols.io.grdbv26 | , , | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC333874/pdf/nar00242-0175.pdf | 1 | 2016 | 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 | 2021-04-15 09:15:21 | ||||
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Rat ACTB one-step qPCR Resource Report Resource Website |
Daniel Groelz, Nadine Dettmann | DOI:10.17504/protocols.io.qnidvce | 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. | Groelz D, Viertler C, Pabst D, Dettmann N, Zatloukal K (2018) Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues. PLoS ONE 13(9): e0203608. doi: 10.1371/journal.pone.0203608 | QIAGEN GmbH, Hilden, Germany, QIAGEN GmbH, Hilden, Germany | https://doi.org/10.1371/journal.pone.0203608 | 1 | 2018 | Daniel Groelz, Nadine Dettmann 2018. Rat ACTB one-step qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.qnidvce | 2021-04-15 09:15:47 | ||
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Epifluorescent Microscopy of Virus Particles Using SYBR Green Resource Report Resource Website |
Steven Wilhelm, Samantha Coy, Alyssa Alsante | DOI:10.17504/protocols.io.qsgdwbw | The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols | 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. | The University of Tennessee, Knoxville, The University of Tennessee, Knoxville, The University of Tennessee, Knoxville | https://doi.org/10.1371/journal.pone.0184371 | 5 | 2018 | 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 | 2021-04-15 09:15:20 | ||
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Generation of DNA fragments by sonication Resource Report Resource Website |
William H. Wilson and Declan Schroeder | DOI:10.17504/protocols.io.dy57y5 | VERVE Net | Manual of Aquatic Viral Ecology | http://www.aslo.org/books/mave/MAVE_134.pdf | 1 | 2016 | William H. Wilson and Declan Schroeder 2016. Generation of DNA fragments by sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.dy57y5 | 2021-04-15 09:15:47 | |||
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immunofluorescence analysis of myosin heavy chain isoforms Resource Report Resource Website |
Momoko Ogitani | DOI:10.17504/protocols.io.iwjcfcn | Daiichi Sankyo Co., Ltd. | 1 | 2017 | Momoko Ogitani 2017. immunofluorescence analysis of myosin heavy chain isoforms. protocols.io https://dx.doi.org/10.17504/protocols.io.iwjcfcn | 2021-04-15 09:15:21 | |||||
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Cultivation / Spore production for Fusarium spp Resource Report Resource Website |
Remco Stam | DOI:10.17504/protocols.io.fmtbk6n | Technische Universiät München | 2 | 2016 | Remco Stam 2016. Cultivation / Spore production for Fusarium spp. protocols.io https://dx.doi.org/10.17504/protocols.io.fmtbk6n | 2021-04-15 09:15:21 | |||||
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Purification of the any avian IgY using chloroform, trichloroacetic acid and affinity purification using HiTrap™ Columns. Resource Report Resource Website |
Angel Justiz-Vaillant | DOI:10.17504/protocols.io.bjnskmee | University of the West Indies, [email protected] | 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. | University of the West Indies St. Augustine | 1 | 2020 | 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 | 2021-04-15 09:15:21 | |||
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In vitro transcription of crRNA and tracrRNA from DNA oligos for cas9 enrichment and nanopore sequencing (for Bac - PULCE) Resource Report Resource Website |
Olin Silander | DOI:10.17504/protocols.io.brbnm2me | 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 | Massey University | 1 | 2021 | 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 | 2021-04-15 09:15:47 |
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