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Name Authors DOI Group Summary Associated Publications RRIDs used Affiliations External URL Version Publication Date Proper Citation Record Last Update
Evaluación la producción de TNF alfa por PBMCs estimulados con LPS tratados con péptidos
 
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Lily Johanna Toro, Germán Alberto Téllez Ramírez, Diana Carolina Henao, Jhon Carlos Castaño Osorio 10.17504/protocols.io.jb6cire Grupo de inmunología molecular Evaluación de la capacidad de neutralización del efecto del Lipopolisacarido (LPS) de inducir la producción de Factor de Necrosis Tumoral (TNFalfa) en Células Mononucleares de Sangre Periférica (PBMCs) tratadas con peptidos cationicos.   Centro de Investigaciones Biomédicas - Universidad del Quindío, Centro de investigaciones biomédicas. Universidad del Quindío., Centro de investigaciones biomédicas. Universidad del Quindío., Centro de investigaciones biomédicas. Universidad del Quindío. 1 2017 Lily Johanna Toro, Germán Alberto Téllez Ramírez, Diana Carolina Henao, Jhon Carlos Castaño Osorio 2017. Evaluación la producción de TNF alfa por PBMCs estimulados con LPS tratados con péptidos. protocols.io dx.doi.org/10.17504/protocols.io.jb6cire 2021-03-29 03:09:36
Ethanol Fixation Protocol
 
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Kelsey Miller 10.17504/protocols.io.e24bggw BioLegend Cell Fixation and Permeabilization Protocol Using 70% Ethanol BioLegend http://www.biolegend.com/media_assets/support_protocol/BioLegend_EthFIXPERM_032912.pdf 1 2016 Kelsey Miller 2016. Ethanol Fixation Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e24bggw 2021-03-29 03:09:36
Preparation of Fungal ITS Amplicons from Soil Samples for Illumina Amplicon Sequencing
 
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Loreen Sommermann, Joerg Geistlinger, Jessica Zwanzig, Ingo Schellenberg 10.17504/protocols.io.nmgdc3w This protocol was used for the molecular profiling of the soil fungal community of two winter wheat fields as a part of a long-term field trial in Germany with following Illumina Amplicon Sequencing. Besides different pre-crops, the effects of soil management and fertilization on the fungal communities were investigated.  Due to different aspects of our protocol, we wanted to ensure reproducing a realistic ratio of the different fungal communities in the soils. The most important steps to conform this demand being the frequently measurements of DNA concentrations at different steps of the protocol, the high numbers of PCRs of each soil sample and the use of the qPCR setting a cut-off for the PCR cycles to avoid an over-amplification of certain PCR-fragments and a loss of detectable biodiversity. The use of the two DNA isolation kits based on the cooperation with our project partner and the exchange of DNAs.  Sommermann L, Geistlinger J, Wibberg D, Deubel A, Zwanzig J, Babin D, Schlüter A, Schellenberg I, Fungal community profiles in agricultural soils of a long-term field trial under different tillage, fertilization and crop rotation conditions analyzed by high-throughput ITS-amplicon sequencing. PLoS ONE 13(4). doi: 10.1371/journal.pone.0195345 Anhalt University of Applied Sciences (AUAS), Institute of Bioanalytical Sciences (IBAS), Bernburg, Anhalt University of Applied Sciences (AUAS), Institute of Bioanalytical Sciences (IBAS), Bernburg, Anhalt University of Applied Sciences (AUAS), Institute of Bioanalytical Sciences (IBAS), Bernburg, Anhalt University of Applied Sciences (AUAS), Institute of Bioanalytical Sciences (IBAS), Bernburg https://doi.org/10.1371/journal.pone.0195345 1 2018 Loreen Sommermann, Joerg Geistlinger, Jessica Zwanzig, Ingo Schellenberg 2018. Preparation of Fungal ITS Amplicons from Soil Samples for Illumina Amplicon Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.nmgdc3w 2021-03-29 03:09:36
Potential Rate Measurements for Uptake of Bicarbonate and Acetate by Microbes in Crustal Fluids
 
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Julie Huber, Caroline Fortunato 10.17504/protocols.io.iutcewn Center for Dark Energy Biosphere Investigations, Huber Lab These were developed to measure potential microbial activity for uptake of acetate and bicarbonate by microbes in venting hydrothermal fluids. The protocol was designed for Axial Seamount diffusely venting fluids, but can be applied to other fluid systems (e.g. cold, oxic fluids, seawater, etc). Concentrations of label will depend on environment of interest. WHOI, WHOI 1 2017 Julie Huber, Caroline Fortunato 2017. Potential Rate Measurements for Uptake of Bicarbonate and Acetate by Microbes in Crustal Fluids. protocols.io dx.doi.org/10.17504/protocols.io.iutcewn 2021-03-29 03:09:36
Single Nuclei RNA Sequencing of Breast Adipose Tissue (10x Nuclei-Seq)
 
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Kevin Nee, Quy Nguyen, Kai Kessenbrock 10.17504/protocols.io.tdwei7e Human Cell Atlas Method Development Community Breast cancer originates in the mammary gland epithelium, however growing evidence demonstrates that the diverse array of stromal tissues influence the behavior of breast epithelium and has key roles in the pathogenesis of breast cancer. Despite increased recognition and research, the heterogeneity of the breast stroma (endothelium, fibroblasts, and adipocytes) poses challenges in elucidating which stromal populations are responsible for the complex interactions of the breast microenvironment. To this end, we have employed single cell RNA sequencing (scRNAseq) of the stromal populations within the breast. However adipose tissue, due to its delicate and lipid filled nature is not amenable to these methods of interrogation. To overcome this obstacle, we have developed a method for isolation of adipose nuclei for 10x sequencing. Together, we will use these approaches to investigate the heterogeneity of stroma and adipocytes, and determine the interactions of the breast microenvironment at single-cell resolution.  University of California-Irvine, Medical Scientist Training Program, School of Medicine, Department of Biological Chemistry, University of California-Irvine, School of Medicine, Department of Biological Chemistry, University of California-Irvine, School of Medicine, Department of Biological Chemistry 1 2018 Kevin Nee, Quy Nguyen, Kai Kessenbrock 2018. Single Nuclei RNA Sequencing of Breast Adipose Tissue (10x Nuclei-Seq) . protocols.io dx.doi.org/10.17504/protocols.io.tdwei7e 2021-03-29 03:09:36
Salmonella blood culture surveillance: Antibiotic susceptibility testing
 
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Sien Ombelet 10.17504/protocols.io.9q9h5z6 Unit of Tropical Bacteriology; Department of Clinical Sciences; Institute of Tropical Medicine Antwerp; Belgium 1 2019 Sien Ombelet 2019. Salmonella blood culture surveillance: Antibiotic susceptibility testing. protocols.io dx.doi.org/10.17504/protocols.io.9q9h5z6 2021-03-29 03:09:36
How to culture corallochytrean Corallochytrium limacisporum, a unicellular relative to animals
 
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Maria Rubio-Brotons 10.17504/protocols.io.gqmbvu6 Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab IBE CSIC-UPF 1 2016 Maria Rubio-Brotons 2016. How to culture corallochytrean Corallochytrium limacisporum, a unicellular relative to animals. protocols.io dx.doi.org/10.17504/protocols.io.gqmbvu6 2021-03-29 03:09:36
Tissue Freezing for seqFISH
 
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Nina Dar, Long Cai 10.17504/protocols.io.7mdhk26 Human BioMolecular Atlas Program (HuBMAP) Method Development Community Once a tissue is received, it should be frozen using the following protocol: California Institute of Technology, California Institute of Technology 1 2019 Nina Dar, Long Cai 2019. Tissue Freezing for seqFISH. protocols.io dx.doi.org/10.17504/protocols.io.7mdhk26 2021-03-29 03:09:39
Preparation of primary chicken embryo liver (CEL) cells
 
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Norfitriah Mohamed Sohaimi, Mohd Hair Bejo, Abdul Rahman Omar, Aini Ideris, Nurulfiza Mat Isa 10.17504/protocols.io.7zchp2w Sohaimi NM, Bejo MH, Omar AR, Ideris A, Isa NM (2019) Molecular characterization of fowl adenovirus isolate of Malaysia attenuated in chicken embryo liver cells and its pathogenicity and immunogenicity in chickens. PLoS ONE 14(12): e0225863. doi: 10.1371/journal.pone.0225863 Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia., Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia., Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia., Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia., Faculty of Biotehnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. https://doi.org/10.1371/journal.pone.0225863 1 2019 Norfitriah Mohamed Sohaimi, Mohd Hair Bejo, Abdul Rahman Omar, Aini Ideris, Nurulfiza Mat Isa 2019. Preparation of primary chicken embryo liver (CEL) cells. protocols.io dx.doi.org/10.17504/protocols.io.7zchp2w 2021-03-29 03:09:39
Pseudomonas syringae seed infections
 
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William Rooney, Janet Laird (University of Glasgow), Mechna Chowdhury (University of Glasgow), Catriona MacIntosh (University of Glasgow), Xiaoyi Deng (University of Glasgow), Phoebe McBride (University of Glasgow), Joel Milner (University of Glasgow) 10.17504/protocols.io.bhwhj7b6 Seed contamination represents a critical route for the transmission of bacterial plant diseases. Here we have created a step-by-step protocol for in vitro seed infections of Pseudomonas syringae. This protocol provides a robust method for P. syringae infection in cucumber. This synthetic seed infection assay is generally applicable for P. syringae as we have successfully adapted it for both tomato and soybean. University of Glasgow, University of Glasgow, University of Glasgow, University of Glasgow, University of Glasgow, University of Glasgow, University of Glasgow 1 2021 William Rooney, Janet Laird (University of Glasgow), Mechna Chowdhury (University of Glasgow), Catriona MacIntosh (University of Glasgow), Xiaoyi Deng (University of Glasgow), Phoebe McBride (University of Glasgow), Joel Milner (University of Glasgow) 2021. Pseudomonas syringae seed infections. protocols.io dx.doi.org/10.17504/protocols.io.bhwhj7b6 2021-03-29 03:09:39
Genotyping of rs3824662 polymorphism in GATA3 gene by allele-specific PCR
 
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Nafiul Huda, Md. Ismail Hosen, Pankaj Kumar Sarkar, A.K.M. Mahbub Hasan, A.H.M. Nurun Nabi 10.17504/protocols.io.k45cyy6 To amplify the target regions of GATA3 for studying rs3824662 polymorphism, a web-based primer designing tool available at http://bioinfo.ut.ee/primer3-0.4.0 facilitates crafting the set of outer primers [forward outer primer: 5’-TTGCAAATGGAAGAGGGTCT-3’ and reverse outer primer: 5’-ACCCTGCAAATGAGAGGAAA-3’] and inner primers [G specific primer: 5’-TGAGATTAAACACAAACACGtTG-3’ and T specific primer: 5’-CTGAGATTAAACACAAACACGaTT-3’] to perform allele-specific PCR that specifically amplify the GATA3 gene. G/T allele-specific primer is very sensitive and binds only with the DNA fragments containing G/T nucleotide template DNA. Forward outer and reverse outer primers will amplify a specific 691 base pair region of GATA3. This 691 base pair region contains the desired polymorphic site of interest. Allele-specific primers produce 506 bp products upon the presence of either a G allele or T allele. PCR reaction in a total volume of 15 μL with an initial denaturing step of 5 min at 95°C, followed by 40 cycles of 30 s at 95°C, 45 s at 55°C, and 1 min at 72°C, and a final extension step of 5 min at 72°C will produce better reproducible result. The reaction mixture contains both forward outer and reverse outer primers and contains either the G allele-specific forward inner primer or the T allele-specific forward inner primer. Primer concentration used was 200 nM each. When the G allele is present in a specific DNA sequence, only the 506 base pair band will be found upon gel electrophoresis of PCR amplicons with the G allele-specific primer. In this case, no such 506 base pair band will be obtained upon gel electrophoresis with the T allele-specific primer. On the other hand, for mutant TT genotypes, the opposite phenomenon will occur when the T allele is present, which will be reflected by the presence of the 506 base pair DNA band. In the case of the heterozygous genotype (GT), both inner primers for G and T alleles will bind with specific DNA sequences to produce 506 base pair bands. Huda N, Hosen MI, Yasmin T, Sarkar PK, Hasan AKMM, Nabi AHMN (2018) Genetic variation of the transcription factor GATA3, not STAT4, is associated with the risk of type 2 diabetes in the Bangladeshi population. PLoS ONE 13(7): e0198507. doi: 10.1371/journal.pone.0198507 University of Dhaka, University of Dhaka, Dinajpur Diabetes O Swasthoseba Hospital, Dinajpur, Bangladesh, University of Dhaka, University of Dhaka https://doi.org/10.1371/journal.pone.0198507 1 2018 Nafiul Huda, Md. Ismail Hosen, Pankaj Kumar Sarkar, A.K.M. Mahbub Hasan, A.H.M. Nurun Nabi 2018. Genotyping of rs3824662 polymorphism in GATA3 gene by allele-specific PCR. protocols.io dx.doi.org/10.17504/protocols.io.k45cyy6 2021-03-29 03:09:39
Radiographic protocol to quantify cranial tibial translation in dogs
 
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Adolfo Maria Tambella, Stefano Martin, Anna Rita Attili, Cecilia Vullo, Luca Omini 10.17504/protocols.io.bc48iyzw This protocol describes a radiographic procedure to objectively quantifyin vivothe cranial tibial translation using a simple device in dogs. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; } This protocol was used in the following publication: .justify:after { content: ""; display:inline-block; width: 100%; } https://doi.org/10.1371/journal.pone.0228621 .justify:after { content: ""; display:inline-block; width: 100%; } Tambella AM, Omini L, Attili AR, Vullo C, Martin S (2020) Evaluation of cranial tibial translation in dogs: Diagnostic accuracy of radiographic method using a simple device. PLoS ONE 15(2): e0228621. (ISSN: 1932-6203) (DOI: 10.1371/journal.pone.0228621) .justify:after { content: ""; display:inline-block; width: 100%; } University of Camerino, School of Biosciences and Veterinary Medicine, Matelica (MC), Italy, University of Camerino, School of Biosciences and Veterinary Medicine, Matelica (MC), Italy, University of Camerino, School of Biosciences and Veterinary Medicine, Matelica (MC), Italy, University of Camerino, School of Biosciences and Veterinary Medicine, Matelica (MC), Italy, Clinica Veterinaria Omini, Chiaravalle (AN), Italy https://doi.org/10.1371/journal.pone.0228621 1 2020 Adolfo Maria Tambella, Stefano Martin, Anna Rita Attili, Cecilia Vullo, Luca Omini 2020. Radiographic protocol to quantify cranial tibial translation in dogs. protocols.io dx.doi.org/10.17504/protocols.io.bc48iyzw 2021-03-29 03:09:40
Applying vContact to Viral Sequences and Visualizing the Output (Cyverse)
 
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Benjamin Bolduc 10.17504/protocols.io.x5xfq7n VERVE Net, Sullivan Lab, iVirus A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses actual reads from Ocean Sampling Day (2014) and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure. The Ohio State University https://doi.org/10.1101/533240 5 2019 Benjamin Bolduc 2019. Applying vContact to Viral Sequences and Visualizing the Output (Cyverse). protocols.io dx.doi.org/10.17504/protocols.io.x5xfq7n 2021-03-29 03:09:41
Protocol of preparation of horseradish peroxidase (HRP) conjugated to anti-human IgG to be used as secondary antibody in immunoassays.
 
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Angel Justiz-Vaillant 10.17504/protocols.io.bjmxkk7n University of the West Indies, [email protected] Peroxidase labeled anti-human IgG is a conjugated secondary antibody that can be used to detect specific antibodies against millions of antigens in humans. This reagent is usually used in enzyme-linked immunosorbent assay, immunoblot analysis and dot blot [1,2]. Reference1. Stubenrauch K, Wessels U, Essig U, Kowalewsky F, Vogel R, Heinrich J. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.J Pharm Biomed Anal. 2013;72:208-215. doi:10.1016/j.jpba.2012.08.0232.Justiz Vaillant AA, McFarlane-Anderson N, Akpaka PE, Smikle MP, Ramirez N, et al. (2013) Use of Dot Blots Analysis in the Separation of Anti-HIV Antibodies in Animals. J Chromat Separation Techniq 4: 181. doi:10.4172/2157- 7064.1000181 University of the West Indies St. Augustine 1 2020 Angel Justiz-Vaillant 2020. Protocol of preparation of horseradish peroxidase (HRP) conjugated to anti-human IgG to be used as secondary antibody in immunoassays. . protocols.io dx.doi.org/10.17504/protocols.io.bjmxkk7n 2021-03-29 03:09:41
Determining Lipid Content in Embryos using Nile Red Fluorescence
 
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Luciano Bonilla, Peter Hansen 10.17504/protocols.io.8qwhvxe University of Florida Bovine Embryo Lab The following protocol is derived from the protocol described by Genicot et al. (Theriogenology 63: 1181- 1194, 2005) and is based on the fluorescence emitted by Nile Red when in association with lipid (see Fowler and Greenspan, J Histochem Cytochem 33: 833-836, 1985). University of Florida, University of Florida https://animal.ifas.ufl.edu/hansen/lab_protocol_docs/detection_lipids_embryos_nile_red_fluorescence.pdf 1 2019 Luciano Bonilla, Peter Hansen 2019. Determining Lipid Content in Embryos using Nile Red Fluorescence. protocols.io dx.doi.org/10.17504/protocols.io.8qwhvxe 2021-03-29 03:09:41
Artificial Cerebrospinal Fluid IX (ACSF.IX)
 
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Allen Institute for Brain Science 10.17504/protocols.io.bdn9i5h6 BICCN, Allen Institute for Brain Science Artificial Cerebrospinal Fluid IX (ACSF.IX) is used for applications including tissue bath solution during electrophysiological recording for probing synaptic connections under physiological conditions.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 IX (ACSF.IX). protocols.io dx.doi.org/10.17504/protocols.io.bdn9i5h6 2021-03-29 03:09:41
Calibration Protocol - Plate Reader Fluorescence Calibration with Fluorescein
 
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Geoff Baldwin, Traci Haddock-Angelli, Jacob Beal, Ari Dwijayanti, Marko Storch, Natalie Farny, Richard Tennant, Paul Rutten 10.17504/protocols.io.548g8zw iGEM Measurement Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve. Although distribution of a known concentration of GFP protein would be an ideal way to standardize the amount of GFP fluorescence in our ​E. coli​ cells, the stability of the protein and the high cost of its purification are problematic. We therefore use the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).You will prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in your plate reader. By measuring these in your plate reader, you will generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration. Before beginning this protocol, ensure that you are familiar with the GFP settings and measurement modes of your instrument. You will need to know what filters your instrument has for measuring GFP, including information about the bandpass width (530 nm / 30 nm bandpass, 25-30 nm width is recommended), excitation (485 nm is recommended) and emission (520-530 nm is recommended) of this filter.Note: The iGEM Abs600 (OD) calibration protocol with microspheres calibration method is a pre-requisite for carrying out this protocol. You will need data from that calibration to analyse the results of this protocol. Imperial College London, iGEM, iGEM Measurement Committee, Imperial College London, Imperial College London, iGEM Measurement Committee, iGEM Measurement Committee, iGEM Measurement Committee https://2019.igem.org/Measurement 2 2019 Geoff Baldwin, Traci Haddock-Angelli, Jacob Beal, Ari Dwijayanti, Marko Storch, Natalie Farny, Richard Tennant, Paul Rutten 2019. Calibration Protocol - Plate Reader Fluorescence Calibration with Fluorescein. protocols.io dx.doi.org/10.17504/protocols.io.548g8zw 2021-03-29 03:09:41
Bystander Receptor Blockade: IFN?R1, TNFRSF1A, ICAM-1 and FAS
 
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Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson 10.17504/protocols.io.hw8b7hw This protocol describes the use of neutralizing antibodies to block receptrs for assessing involvement in bystander killing by BiTE®-activated T cells. Ross SL, Sherman M, McElroy PL, Lofgren JA, Moody G, Baeuerle PA, Coxon A, Arvedson T, Bispecific T cell engager (BiTE) antibody constructs can mediate bystander tumor cell killing. PLoS ONE 12(8). doi: 10.1371/journal.pone.0183390 Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc., Department of Oncology Research, Amgen Inc. https://doi.org/10.1371/journal.pone.0183390 1 2017 Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson 2017. Bystander Receptor Blockade: IFN?R1, TNFRSF1A, ICAM-1 and FAS. protocols.io dx.doi.org/10.17504/protocols.io.hw8b7hw 2021-03-29 03:09:39
Protocol
 
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Miyuki Takasu 10.17504/protocols.io.bavbie2n Takasu M, Kondo S, Akiyama Y, Takahashi Y, Maeda S, Baba Y, Kawase T, Ichinohe T, Awai K (2020) Assessment of early treatment response on MRI in multiple myeloma: Comparative study of whole-body diffusion-weighted and lumbar spinal MRI. PLoS ONE 15(2): e0229607. doi: 10.1371/journal.pone.0229607 Department of Diagnostic Radiology, Graduate School of Biomedical Sciences, Hiroshima University https://doi.org/10.1371/journal.pone.0229607 1 2019 Miyuki Takasu 2019. Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bavbie2n 2021-03-29 03:09:39
Centrifuge Report of Normal vs Dermatitis Skin Microbiome
 
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James Thornton Jr. 10.17504/protocols.io.g9gbz3w Hurwitz Lab, iMicrobeOutreach Centrifuge is a classification software used on whole genome shotgun (WGS) reads. It uses a reference database to determine what organisms are found in your sample. This protocol will show you how to access the data on CyVerse and walk you through some questions to help in the analysis of the plots.  Hurwitz Lab 1 2017 James Thornton Jr. 2017. Centrifuge Report of Normal vs Dermatitis Skin Microbiome. protocols.io dx.doi.org/10.17504/protocols.io.g9gbz3w 2021-03-29 03:09:39

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