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Extracellular DNA extraction Resource Report Resource Website |
Charline Giguet-Covex, Pierre Taberlet, Francesco Gentile Ficetola | 10.17504/protocols.io.bdwsi7ee | Over the past decade, an increasing number of studies has used environmental DNA from lake sediments to trace past lake ecosystem and landscape changes, agricultural activities or human presence and more broadly the biodiversity. In the environment, DNA can be found as intracellular and extracellular DNA (iDNA and exDNA). The contribution of each of these pools varies according to the environments, but exDNA often represents a high proportion of the total DNA (e.g. Vuillemin et al. 2017 and reference therein). Focusing the analyses on these different pools will lead to different community composition and structure of communities (e.g. Vuillemin et al. 2017). For plants, we propose to focus on the exDNA fraction to avoid the extraction of DNA from plant macro-remains, which might lead to an overrepresentation of these taxa and limit the detection of the other, “rarer” taxa. The manipulation of ancient DNA is delicate, and the biases brought during the experiments can be multiple. Therefore, it is essential to work carefully, under strict laboratory conditions, with multiple controls and several replicates of samples or extraction or PCR (e.g. Fulton 2012).This protocol details a sampling and extraction method of exDNA from sediments. This method was firstly developed for soils by Taberlet et al. 2012. It uses a phosphate buffer to desorb the DNA fragments from particles such as clays and then, the binding, wash and elution buffers from the NucleoSpin® Soil kit (Macherey-Nagel). Here we present a modified protocol from Taberlet et al. 2012. One modification consists in the addition of a concentration step (by using the amicon ultra centrigugal filter system) after the mixing of sediments with the saturated phosphate buffer. This step allow to increase the DNA yield. The quantity of phosphate buffer is usually based on the quantity of wet sediments (e.g. for 15g of wet sediment/soil, we add 15 ml of phosphate buffer). However, because lake sediments can have very different water content (depending on the composition and compaction) and because the exDNA is adsorbed onto particles, we now propose to base the phosphate buffer quantity on the dry weight of sediments. Fulton T., 2012. Setting Up an Ancient DNA Laboratory. Book Chapter, Ancient DNA in Methods in Molecular Biology edited by Shapiro B. and Hofreiter M., 1-11. Taberlet, P., Prud’Homme, S.M., Campione, E., Roy, J., Miquel, C., Shehzad, W., Gielly, L., Rioux, D., Choler, P., Clément, J.‐C., Melodelima, C., Pompanon, F. and Coissac, E, 2012. Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies. Molecular Ecology, 21: 1816-1820. Vuillemin A., Horn F., Alawi M., Henny C., Wagner D., Crowe S. A., Kallmeyer J., 2017. Preservation and Significance of Extracellular DNA in Ferruginous Sediments from Lake Towuti, Indonesia. Frontiers in Microbiology 8, 1440. | [Laboratoire Environnements, DYnamiques et TErritoires de la Montagne (EDYTEM), Université Savoie Mont Blanc, F-73376 Le Bourget du Lac, France], [Laboratoire d’Ecologie Alpine (LECA), Université Grenoble Alpes, F-38000 Grenoble, France], [Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, via Celoria 26, I- 20133 Milano, Italy; Laboratoire d’Ecologie Alpine (LECA), Université Grenoble Alpes, F-38000 Grenoble, France] | 1 | 2020 | Charline Giguet-Covex, Pierre Taberlet, Francesco Gentile Ficetola 2020. Extracellular DNA extraction. protocols.io dx.doi.org/10.17504/protocols.io.bdwsi7ee | 2021-03-29 03:08:57 | ||||
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Heterologous protein expression in E. coli Resource Report Resource Website |
Diep Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan | 10.17504/protocols.io.bdjti4nn | Pogson Genomics Group | Protocol for recombinant protein expression in E. coli for protein purification and subsequent enzyme assays, protein crystallography etc. | The Australian National University, The Australian National University, The Australian National University, The Australian National University, The Australian National University | 5 | 2020 | Diep Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan 2020. Heterologous protein expression in E. coli. protocols.io dx.doi.org/10.17504/protocols.io.bdjti4nn | 2021-03-29 03:08:26 | |||
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Astrios EQ instrument setup and sample acquisition Resource Report Resource Website |
Aizea Morales-Kastresana, Joshua Welsh, Jennifer Jones | 10.17504/protocols.io.bj6tkren | Translational Nanobiology Section | In this protocol the authors describe the general steps to follow in order to achieve optimallaser:stream:detectors alignment and sample acquisition on the Astrios EQ flow cytometer (Beckman Coulter). The general concept however can be applied to other jet-in-air instruments. | Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health | 1 | 2020 | Aizea Morales-Kastresana, Joshua Welsh, Jennifer Jones 2020. Astrios EQ instrument setup and sample acquisition . protocols.io dx.doi.org/10.17504/protocols.io.bj6tkren | 2021-03-29 03:08:26 | |||
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Stacking gel buffer (4X) Resource Report Resource Website |
DAVID DUNIGAN AND IRINA AGARKOVA | 10.17504/protocols.io.eq4bdyw | VERVE Net | For use in Polyacrylamide Gel System For Electrophoresis Of Proteins. | 1 | 2016 | DAVID DUNIGAN AND IRINA AGARKOVA 2016. Stacking gel buffer (4X). protocols.io dx.doi.org/10.17504/protocols.io.eq4bdyw | 2021-03-29 03:08:26 | ||||
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Bacterial transformation Resource Report Resource Website |
Laura Sánchez | 10.17504/protocols.io.6taheie | AEGIS - Madrid iGEM 2019 | Transformation of competent Escherichia coli cultures with heterologous DNA. This protocol allows the introduction of external DNA into competent E. coli cells. Typically, the piece of DNA consists of a plasmid, either for its cloning or for heterologous protein expression. | AEGIS - Madrid iGEM Team 2019 | https://www.addgene.org/protocols/bacterial-transformation/ | 1 | 2019 | Laura Sánchez 2019. Bacterial transformation. protocols.io dx.doi.org/10.17504/protocols.io.6taheie | 2021-03-29 03:08:28 | ||
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Japanese encephalitis virus real-time RT-PCR Resource Report Resource Website |
Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay | 10.17504/protocols.io.kr9cv96 | Public Health Virology, Forensic and Scientific Services | A real-time RT-PCR using an MGB probe, this assay detects Japanese encephalitis virus (JEV) from human and mosquito samples.The assay targets the 3'UTR region of known JEV strains. | Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services, Public Health Virology, Forensic and Scientific Services | https://doi.org/10.1089/vbz.2013.1373 | 2 | 2017 | Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay 2017. Japanese encephalitis virus real-time RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.kr9cv96 | 2021-03-29 03:08:28 | ||
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Drug tracking on hydra Resource Report Resource Website |
Ida Barlow, Adam Mcdermott-Rouse, Luigi Feriani | 10.17504/protocols.io.8z4hx8w | Behavioural Genomics | Protocol for preparing worms, preparing drug plates, dispensing worms with wormsorter and tracking on Hydra rigs | Imperial College London, Imperial College London, Imperial College London | 1 | 2019 | Ida Barlow, Adam Mcdermott-Rouse, Luigi Feriani 2019. Drug tracking on hydra. protocols.io dx.doi.org/10.17504/protocols.io.8z4hx8w | 2021-03-29 03:08:28 | |||
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MojoSort™ Human anti-APC Nanobeads Column Protocol Resource Report Resource Website |
Sam Li | 10.17504/protocols.io.7afhibn | BioLegend | 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.Note: Due to the properties of our beads, it may be possible to use far fewer beads than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed. | BioLegend | https://www.biolegend.com/protocols/mojosort-human-anti-apc-nanobeads-column-protocol/4758/ | 1 | 2019 | Sam Li 2019. MojoSort™ Human anti-APC Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7afhibn | 2021-03-29 03:08:28 | ||
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Chlorovirus DNA Miniprep Procedure Resource Report Resource Website |
David Dunigan and Irina Agarkova | 10.17504/protocols.io.erzbd76 | VERVE Net | The University of Nebraska-Lincoln | 1 | 2016 | David Dunigan and Irina Agarkova 2016. Chlorovirus DNA Miniprep Procedure. protocols.io dx.doi.org/10.17504/protocols.io.erzbd76 | 2021-03-29 03:08:28 | ||||
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Hepatorenal index protocol Resource Report Resource Website |
fabio10stahl , Fabio Lucio Stahlschmidt | 10.17504/protocols.io.bq2jmycn | Radiochir Gruppe | Pontificia Universidade Católica do Paraná, Pontificia Universidade Católica do Paraná | 1 | 2020 | fabio10stahl , Fabio Lucio Stahlschmidt 2020. Hepatorenal index protocol. protocols.io dx.doi.org/10.17504/protocols.io.bq2jmycn | 2021-03-29 03:08:28 | ||||
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TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol Resource Report Resource Website |
Sam Li | 10.17504/protocols.io.8aahsae | BioLegend | Buyer is solely responsible for determining whether Buyer has all intellectual property rights that are necessary for Buyer's intended uses of the BioLegend TotalSeq™ products. For example, for any technology platform Buyer uses with TotalSeq™, it is Buyer's sole responsibility to determine whether it has all necessary third party intellectual property rights to use that platform and TotalSeq™ with that platform. TotalSeq™-B antibodies are designed to be used with 10x Single Cell 3’ Reagent Kit v3 with Feature Barcoding. The adjusted protocol below is for customers who are utilizing TotalSeq™-A antibodies with the v3 kit instead. Note: Step 4 from the 10x Genomics user guide document number CG000185, Rev B only applies to the cDNA library and NOT to the TotalSeq™-A library. Please read the entire protocol below and the 10x Genomics user guide for the Chromium Single Cell 3ʹ Reagent Kits v3 with Feature Barcoding technology for Cell Surface Protein before starting the experiments. 10x Genomics user guide document number CG000185, Rev B. Commonly used abbreviations:ADT: Antibody derived tags, the oligo sequence conjugated to regular TotalSeq™-A antibodiesHTO: Hashtag oligonucleotides, the oligo sequence conjugated to TotalSeq™-A Hashtag antibodies | BioLegend | https://www.biolegend.com/protocols/totalseq-a-antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-31-protocol/5010/ | 1 | 2019 | Sam Li 2019. TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol. protocols.io dx.doi.org/10.17504/protocols.io.8aahsae | 2021-03-29 03:08:27 | ||
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Three-dimensional computerized forensic craniofacial reconstruction (CCFR) Resource Report Resource Website |
Geraldo Elias Miranda, Caroline Wilkinson, Mark Roughley, Thiago Leite Beaini, Rodolfo Francisco Haltenhoff Melani | 10.17504/protocols.io.m4xc8xn | A description of to how to produce and evaluate three-dimensional computerized forensic craniofacial reconstructions (CCFR) using Horos, Blender, Cloud Compare, MakeHuman and Picassa computer programs. | Universidade Federal de São Paulo, Liverpool John Moores University, Liverpool John Moores University, Universidade Federal de Uberlândia, Universidade Federal de São Paulo | 1 | 2018 | Geraldo Elias Miranda, Caroline Wilkinson, Mark Roughley, Thiago Leite Beaini, Rodolfo Francisco Haltenhoff Melani 2018. Three-dimensional computerized forensic craniofacial reconstruction (CCFR). protocols.io dx.doi.org/10.17504/protocols.io.m4xc8xn | 2021-03-29 03:08:27 | ||||
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Identifying Viral Sequences Using VirSorter (Cyverse) Resource Report Resource Website |
Benjamin Bolduc | 10.17504/protocols.io.eyjbfun | Sullivan Lab, iVirus | Identifying putative viral sequences from SPAdes-assembled data from the Ocean Sampling Day (2014) metagenomic datasets using VirSorter. | Bolduc B, Youens-Clark K, Roux S, Hurwitz BL, Sullivan MB, iVirus: facilitating new insights in viral ecology with software and community data sets imbedded in a cyberinfrastructure. The ISME Journal 11(1). doi: ismej201689 | The Ohio State University | https://www.nature.com/articles/ismej201689.pdf?origin=ppub | 2 | 2016 | Benjamin Bolduc 2016. Identifying Viral Sequences Using VirSorter (Cyverse). protocols.io dx.doi.org/10.17504/protocols.io.eyjbfun | 2021-03-29 03:08:28 | |
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CviJI Buffer B Resource Report Resource Website |
DAVID DUNIGAN AND IRINA AGARKOVA | 10.17504/protocols.io.esjbecn | VERVE Net | For use in CviJI Purification From IL-3A Virus Infected NC64A Chlorella. | 1 | 2016 | DAVID DUNIGAN AND IRINA AGARKOVA 2016. CviJI Buffer B. protocols.io dx.doi.org/10.17504/protocols.io.esjbecn | 2021-03-29 03:08:27 | ||||
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Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing Resource Report Resource Website |
Julie Haendiges, Ruth Timme, Maria Balkey | 10.17504/protocols.io.bja6kihe | GenomeTrakr | This procedure outlines the procedures for operation and maintenance of an Illumina MiSeq Sequencer for Whole Genoem Sequencing.This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network.Complete in order:1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube)Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing2. DNA QuantitationQuantitation of extracted DNA using the Qubit Flourometer 3. Library Preparation for WGS (Library Preparation using Illumina DNA Prep or Library Preparation using Illumina Nextera XT )Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex)2. DNA QuantitationQuantitation of extracted DNA using the Qubit Flourometer 3. Library Preparation for WGS (Library Preparation using Illumina DNA Prep or Library Preparation using Illumina Nextera XT )Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex)3. Library Preparation for WGS (Library Preparation using Illumina DNA Prep or Library Preparation using Illumina Nextera XT )4. Sequencing using Illumina MiSeq (Included SOP)5. Data Quality Checks and NCBI Submission | US Food and Drug Administration, US Food and Drug Administration, US Food and Drug Administration | 1 | 2020 | Julie Haendiges, Ruth Timme, Maria Balkey 2020. Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.bja6kihe | 2021-03-29 03:08:27 | |||
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Adipose depot innervation: whole mount staining, imaging, quantification Resource Report Resource Website |
Kristy Townsend, Jake Willows | 10.17504/protocols.io.6nzhdf6 | Townsend Lab Neurobiology & Energy Balance | Abstract The difficulty in obtaining as well as maintaining weight loss, together with the impairment of metabolic control in conditions like diabetes and cardiovascular disease, may represent pathological situations of inadequate neural communication between the brain and peripheral organs and tissues. Innervation of adipose tissues by peripheral nerves provides a means of communication between the master metabolic regulator in the brain (chiefly the hypothalamus), and energy-expending and energy-storing cells in the body (primarily adipocytes). Although chemical and surgical denervation studies have clearly demonstrated how crucial adipose tissue neural innervation is for maintaining proper metabolic health, we have uncovered that adipose tissue becomes neuropathic (ie: reduction in neurites) in various conditions of metabolic dysregulation. Here, utilizing both human and mouse adipose tissues, we present evidence of adipose tissue neuropathy, or loss of proper innervation, under pathophysiological conditions such as obesity, diabetes, and aging, all of which are concomitant with insult to the adipose organ as well as metabolic dysfunction. Neuropathy is indicated by loss of nerve fiber protein expression, reduction in synaptic markers, and lower neurotrophic factor expression in adipose tissue. Aging-related adipose neuropathy particularly results in loss of innervation around the tissue vasculature, which cannot be reversed by exercise. Together with indications of neuropathy in muscle and bone, these findings underscore that peripheral neuropathy is not restricted to classic tissues like the skin of distal extremities, and that loss of innervation to adipose may trigger or exacerbate metabolic diseases. In addition, we have demonstrated stimulation of adipose tissue neural plasticity with cold exposure, which may ameliorate adipose neuropathy and be a potential therapeutic option to re-innervate adipose and restore metabolic health. | Ohio State University, Ohio State University, Columbus | 1 | 2019 | Kristy Townsend, Jake Willows 2019. Adipose depot innervation: whole mount staining, imaging, quantification. protocols.io dx.doi.org/10.17504/protocols.io.6nzhdf6 | 2021-03-29 03:08:27 | |||
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Quantification of plant hormones by standard addition method Resource Report Resource Website |
Takuyu Hashiguchi, Masatsugu Hashiguchi, Hidenori Tanaka, Koki Fukushima, Takahiro Gondo, Ryo Akashi | 10.17504/protocols.io.bqy6mxze | Phytohormones are essential signaling molecules in multiple plant processes, including growth, development, and stress response. Simultaneous quantification of multiple plant hormones is required for understanding plant physiology because hormones typically change in tandem in response to external stimuli. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) adopts stable isotope-labeled compounds in previously published works. However, a method for quantifying phytohormones when isotopically labeled chemicals are not available, remains to be established.This protocol can be used for measuring plant hormone content by standard addition method. Standard addition method has been used for quantification of various compounds such as drugs and pesticides especially when it is difficult to obtain stable isotope-labeled compounds. Plant hormones are extracted from a plant material such as Lotus japonicus with 50% acetonitrile and partially purified with reversed-phase solid-phase extraction (SPE) and ready for LC-MS/MS analysis. Calibration curves are constructed by spiking pure standards to the matrix. The method allows us to quantify plant hormones in plant tissues of interest by correcting matrix effects. | Hashiguchi T, Hashiguchi M, Tanaka H, Fukushima K, Gondo T, Akashi R (2021) Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method. PLoS ONE 16(2): e0247276. doi: 10.1371/journal.pone.0247276 | Department of Animal and Grassland Sciences, University of Miyazaki, Department of Animal and Grassland Sciences, University of Miyazaki, Department of Animal and Grassland Sciences, University of Miyazaki, Department of Animal and Grassland Sciences, University of Miyazaki, Department of Animal and Grassland Sciences, University of Miyazaki, Department of Animal and Grassland Sciences, University of Miyazaki | https://doi.org/10.1371/journal.pone.0247276 | 1 | 2021 | Takuyu Hashiguchi, Masatsugu Hashiguchi, Hidenori Tanaka, Koki Fukushima, Takahiro Gondo, Ryo Akashi 2021. Quantification of plant hormones by standard addition method. protocols.io dx.doi.org/10.17504/protocols.io.bqy6mxze | 2021-03-29 03:08:27 | ||
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PlOSONE folder Resource Report Resource Website |
Sung-Jin Bae, Inah Kim, Jaechul Song, Euy-Suk Chung | 10.17504/protocols.io.bd3xi8pn | BackgroundThis study investigated the efficacy of first-generation (cefazolin) and third-generation (ceftizoxime) prophylactic antibiotics in patients undergoing cardiac surgery and the incidence of surgical site infections, hospital stay lengths, and medical costs.MethodsAll adult patients (≥20 years) undergoing cardiac surgery (coronary artery bypass surgery, valve operation, or combined surgery) at one hospital from January 01, 2009 to December 31, 2016 were included in this study. A single prophylactic antibiotic was administered at a dose of 1 g within 1 hour of surgical incision and for three days after surgery at eight-hour intervals. After the propensity score matching, 194 patients in each antibiotic prophylaxis groups (first-generation vs third-generation) were analyzed. Among the 388 patients, the incidence of surgical site infection were compared according to the type of prophylactic antibiotics and risk factors were evaluated by chi-squared tests followed by multivariate logistic regression analysis. A Student’s t-tests were analyzed to compare hospitalization and medical costs. ResultsThe incidence of deep surgical site infections significantly lower in first-generation group (5.7%) than third-generation group (16.5%). The pathogens isolated from surgical infection sites were similarly distributed in both groups, but gram-positive bacteria were more highly infectious than gram-negative bacteria (67% vs 23%). Preoperative hospitalization duration, mean operation time, and ventilator use time were similar in both groups but the postoperative hospitalization duration was significantly shorter in the first-generation group (25.5 days) than third-generation (29.8 days). In addition, the medical cost lower in the first-generation group (20,594 USD) than third-generation (26,488 USD).ConclusionIn conclusion, the first-generation (cefazolin) is better than the third-generation (ceftizoxime) as a prophylactic antibiotic in reducing surgical site infection rates, hospitalization lengths, and medical expenditures. | Hanyang University Graduate School, Department of Occupational and Environment Medicine, Hanyang University College of Medicine, Seoul, Korea, Department of Occupational and Environment Medicine, Hanyang University College of Medicine, Seoul, Korea, Department of Cardiovascular surgery, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, Koreal | https://journals.plos.org/plosone/ | 1 | 2020 | Sung-Jin Bae, Inah Kim, Jaechul Song, Euy-Suk Chung 2020. PlOSONE folder. protocols.io dx.doi.org/10.17504/protocols.io.bd3xi8pn | 2021-03-29 03:08:27 | |||
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Semi-thin section analysis Resource Report Resource Website |
Sue Lin | 10.17504/protocols.io.zz5f786 | Lin S, Miao Y, Su S, Xu J, Jin L, Sun D, Peng R, Huang L, Cao J (2019) Comprehensive analysis of Ogura cytoplasmic male sterility-related genes in turnip (Brassica rapa ssp. rapifera) using RNA sequencing analysis and bioinformatics. PLoS ONE 14(6): e0218029. doi: 10.1371/journal.pone.0218029 | Institute of Life Sciences, Wenzhou University, Wenzhou, China | https://doi.org/10.1371/journal.pone.0218029 | 1 | 2019 | Sue Lin 2019. Semi-thin section analysis. protocols.io dx.doi.org/10.17504/protocols.io.zz5f786 | 2021-03-29 03:08:27 | |||
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Electrophysiological recording of electrically-evoked compound action potentials Resource Report Resource Website |
James Fallon, Sophie Payne | 10.17504/protocols.io.bfwyjpfw | SPARC | Recording electrically-evoked compound action potentials (ECAPs) is performed to confirm that the electrode array is appropriately placed along the nerve. Recording ECAPS also allows confirmation that electrical stimulation is above neural threshold i.e. that electrical stimulation activates neural activity. The procedure is performed under anesthesia (immediately post-surgery) or in awake animals, and the procedure should incorporate all local requirements for standards of animal experimentation. | Bionics Institute, Bionics Institute | 1 | 2020 | James Fallon, Sophie Payne 2020. Electrophysiological recording of electrically-evoked compound action potentials. protocols.io dx.doi.org/10.17504/protocols.io.bfwyjpfw | 2021-03-29 03:08:30 |
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