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Authors: Jeyanthi Suppiah
Summary: 1)The partial E gene of dengue virus is amplified before sequencing by using four sets of serotype-specific    oligonucleotides as referenced in the manuscript text.2) The reaction mixture is prepared as the following:    12.5ul of MyTaq RT-PCR (Bioline, Korea)      4.5ul of  Nuclease-Free Water      1.0 ul of Foward Primer for the respective serotype  (10uM)      1.0 ul of Reverse Primer for the respective serotype (10uM)      0.5ul of RNase inhibitor      0.5ul of Reverse Transcriptase (RT) 3) Aliquot 20ul in each PCR tubes. 4) 5ul of Dengue RNA (with known serotype) is added to the corresponding PCR tube. 5) PCR amplification is performed on CFX-96 (BioRad) conventional PCR thermal cycler. 6) The PCR is performed with the following cycling profile: 35 cycles (94 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min) followed by an extension reaction at        72 °C for 5 min. 7) A 25 µl aliquot of each PCR reaction is analyzed on 1.5 % pre-stained agarose by gel electrophoresis, run for 30 min at 90 V. 8) The gel is then viewed under UV illumination. 9) The expected amplicon size for each serotype is 578bp for DENV1, 617bp for DENV 2, 582bp for DENV3 and 572bp for DENV 4 The corresponding      amplicons are extracted from the agarose gel and purified by Gel Extraction Kit (Qiagen, USA) according to the manufacturer ’s instruction. 10) The final elution volume is 30 µl of purified PCR amplicons. Then, 5 µl of these are reanalyzed on 1.5 % agarose gel to substantiate the accuracy of       purification step. The purified PCR amplicons were outsourced for sequencing.

Proper citation: Jeyanthi Suppiah 2018. Dengue genotyping by E gene amplification and sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.racd2aw Copy   

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Authors: Amany Elshafee, Annika Karch, Kristina I. Ringe, Hoen-oh Shin, Hans-Jürgen Raatschen, Nermin Yehia Soliman, Frank Wacker, Jens Vogel-Claussen
Summary: Abstract: .justify:after { content: ""; display:inline-block; width: 100%; } Objectives: To evaluate frequency and severity of complications after CT-guided lung biopsy using the Society of Interventional Radiology (SIR) classification, and to assess risk factors for overall and major complications. .justify:after { content: ""; display:inline-block; width: 100%; } Methods: 311 consecutive biopsies with a non-coaxial semi-automated 18 gauge biopsy system were retrospectively evaluated. Complications after biopsy were classified into minor SIR1-2 and major SIR3-6. Studied risk factors for complications were patient-related (age, sex and underlying emphysema), lesion-related (size, location, morphologic characteristics, depth from the pleura and histopathology), and technique-related (patient position during procedure, thoracic wall thickness at needle path, procedure time length and number of procedural CT images, number of pleural passes, fissure penetration and needle-to-blood vessel angle). Data were analyzed using logistic and ordinal regression. .justify:after { content: ""; display:inline-block; width: 100%; } Results: Complications were pneumothorax and pulmonary hemorrhage. The complications were minor SIR1-2 in 142 patients (45.6%), and major SIR3-4 in 25 patients (8%). SIR5-6 complications were not present. Emphysema, smaller deeply located lesion, increased puncture time length and number of procedural CT images, multiple pleural passes and fissure puncture were significant risk factors for complications severity in univariate analysis. Emphysema (OR=8.8, p .justify:after { content: ""; display:inline-block; width: 100%; } Conclusions: Knowledge about risk factors influencing complications severity is important for planning and performing CT-guided lung biopsies. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Amany Elshafee, Annika Karch, Kristina I. Ringe, Hoen-oh Shin, Hans-Jürgen Raatschen, Nermin Yehia Soliman, Frank Wacker, Jens Vogel-Claussen 2019. Complications of CT-guided lung biopsy with a non-coaxial semi-automated 18 gauge biopsy system: frequency, severity and risk factors. protocols.io https://dx.doi.org/10.17504/protocols.io.wvrfe56 Copy   

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Authors: Laura Bugada, Simon Blanchoud
Group: Blanchoud lab, UNIFR
Summary: We have been subcloning colonial ascidians since some time already, till now - with a lot of success. This protocol has been ruinously used by many of our co-workers.

Proper citation: Laura Bugada, Simon Blanchoud 2021. Subcloning colonial ascidians. protocols.io https://dx.doi.org/10.17504/protocols.io.brw9m7h6 Copy   

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Authors: CAN-ASC Canadian Autoimmunity Standardization Core

Proper citation: CAN-ASC Canadian Autoimmunity Standardization Core 2021. CAN-ASC CONSENSUS PROTOCOL: Isolation, Cryopreservation and Thawing of Peripheral Blood Mononuclear Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.brrsm56e Copy   

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Authors: Ken Christensen
Summary: The Thermo Scientific HisPur Ni-NTA Resin enables effective immobilized metal affinity chromatography (IMAC) purification of polyhistidine-tagged proteins from a soluble protein extract. This resin is composed of nickel-charged nitrilotriacetic acid (NTA) chelate immobilized onto 6% crosslinked agarose. The Ni-NTA resin is compatible with native or denaturing conditions and can be used in multiple formats, including conventional gravity-flow chromatography, spin column and FPLC. Ni-NTA resins are commonly chosen for His-tagged-protein purification because of the four metal-binding sites on the chelate, which allow for high-binding capacity and low-metal ion leaching.

Proper citation: Ken Christensen 2020. HisPur Purification of His-Tagged Proteins--CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.bmg7k3zn Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 10 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults > 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.

Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Quality Assurance and Quality Control (Part 10 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj57kq9n Copy   

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Authors: Julius Judd, Luke A. Wojenski, Lauren M. Wainman, Nathaniel D. Tippens, Edward J. Rice, Alexis Dziubek, Geno J. Villafano, Erin M. Wissink, Philip Versluis, Lina Bagepalli, Sagar R. Shah, Dig B. Mahat, Jacob M. Tome, Charles G. Danko, John T. Lis, Leighton J. Core

Proper citation: Julius Judd, Luke A. Wojenski, Lauren M. Wainman, Nathaniel D. Tippens, Edward J. Rice, Alexis Dziubek, Geno J. Villafano, Erin M. Wissink, Philip Versluis, Lina Bagepalli, Sagar R. Shah, Dig B. Mahat, Jacob M. Tome, Charles G. Danko, John T. Lis, Leighton J. Core 2020. A rapid, sensitive, scalable method for Precision Run-On sequencing (qPRO-seq). protocols.io https://dx.doi.org/10.17504/protocols.io.57dg9i6 Copy   

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Authors: Anne Vogel, Martin Hohmann-Marriott, Rahmi Lale
Group: PhotoSynLab, NTNU Biotechnology
Summary: This protocol discribes how to make growth medium for Synechococcus sp. PCC 7002, including the stock solutions needed.

Proper citation: Anne Vogel, Martin Hohmann-Marriott, Rahmi Lale 2018. Making AA+ medium for Synechococcus sp. PCC 7002. protocols.io https://dx.doi.org/10.17504/protocols.io.kg3ctyn Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. They have both paracrine and autocrine function. Interleukins are also used in animal studies to investigate aspect related to clinical medicine.[1]T cells produce IL-2. The principal targets are T cells. Its primary effects are T-cell proliferation and differentiation, increased cytokine synthesis, potentiating Fas-mediated apoptosis, and promoting regulatory T cell development. It causes proliferation and activation of NK cells and B-cell proliferation and antibody synthesis. Also, it stimulates the activation of cytotoxic lymphocytes and macrophages. .[1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-2 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj22kqge Copy   

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Authors: Jenna Clark, Melanie C. Green, Joseph J. P. Simons
Summary: Persuasion research often focuses on how source characteristics affect attitude change in response to a message; however, message characteristics may also alter perceptions of the source. The Message-Based Impression Formation effect (M-BIF) suggests that perceivers use features of messages to infer characteristics of the source, and that such inferences may have a variety of consequential outcomes. In particular, the choice of narrative versus statistical evidence may have implications for the perceived warmth and competence of a source. In five experiments, narrative arguments led to greater perceptions of source warmth and statistical arguments led to greater perceptions of source competence. Across the two behavioral studies, a matching effect emerged: participants preferred to work on cooperative tasks with partners who had provided narratives, and competitive tasks with partners who had provided statistical evidence. These results suggest that the evidence type chosen for everyday communications may affect person perception and interpersonal interaction.

Proper citation: Jenna Clark, Melanie C. Green, Joseph J. P. Simons 2019. Beyond Persuasion: Evidence Type Affects Impressions of a Message Source. protocols.io https://dx.doi.org/10.17504/protocols.io.7rahm2e Copy   

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Authors: Sven Van Poucke, Dana Huskens, Kurt Van der Speeten, Mark Roest, Bart Lauwereins, Ming-Hua Zheng, Seppe Dehaene, Joris Penders, Abraham Marcus, Marcus Lancé
Group: HIPACAT
Summary: Standard hematological analyses EDTA-anticoagulated blood was used for cytometric analysis using a whole blood counter Sysmex XE 2100® (Sysmex,Kobe, Japan) to obtain a whole blood count. Fibrinogen levels were determined with an ACL-9000 (Diamond Diagnostics, Holliston, MA) coagulation analyser, using a PT-fibrinogen high sensitivity reagent, which is a high-sensitivity calcium thromboplastin that allows the simultaneous determination of PT and fibrinogen levels. aPTT was measured using an ACL-9000 coagulation analyser and INR was calculated by the formula INR (PT patient/PT normal).

Proper citation: Sven Van Poucke, Dana Huskens, Kurt Van der Speeten, Mark Roest, Bart Lauwereins, Ming-Hua Zheng, Seppe Dehaene, Joris Penders, Abraham Marcus, Marcus Lancé 2017. Standard hematological analyses HIPEC . protocols.io https://dx.doi.org/10.17504/protocols.io.ki6cuhe Copy   

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Authors: Dr. Steven Wilhelm, Alyssa Alsante
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.

Proper citation: Dr. Steven Wilhelm, Alyssa Alsante 2017. Running the Thermo Scientific Arktik Thermal Cycler. protocols.io https://dx.doi.org/10.17504/protocols.io.in5cdg6 Copy   

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Authors: Christian Lawrence, Jason Best, Althea James, Shane Hurley, Mitchel Shia, Michelle Urh, Brady Hirshfeld, Nina Bakker, Hugo Perdomo, Aaron Krueger
Summary: Environmental and housing conditions experienced by laboratory animals exert profound effects on their biology, physiology, and behavior.  These parameters are important and often overlooked sources of potential variation in experiments, and should be reported in peer-reviewed publications in order to promote scientific reproducibility.  To that end, here we provide a summary of the environmental conditions in zebrafish (Danio rerio) aquaculture facilities at Boston Children's Hospital (BCH).   We include data on the physico-chemical, health, and nutrition of zebrafish in three separate facilities at BCH: Karp, Enders SSB, and Enders Lobby. In this year's version, we also include new information on our sanitization protocol validation, and dietary inputs. We also provide some additional characterization of our sampling methods for water parameters and health in an updated legend. This information will be applicable to any study involving zebrafish conducted in one of these three facilities during the year of 2019.

Proper citation: Christian Lawrence, Jason Best, Althea James, Shane Hurley, Mitchel Shia, Michelle Urh, Brady Hirshfeld, Nina Bakker, Hugo Perdomo, Aaron Krueger 2020. Zebrafish (Danio rerio) Environmental Summary, Aquatic Resources Program, Boston Children's Hospital 2019. protocols.io https://dx.doi.org/10.17504/protocols.io.bb2iiqce Copy   

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Authors: Linda Thomas, Angie Brown, Kerry Wiles
Group: Laboratory of Systems Pharmacology, NCIHTAN
Summary: This procedure establishes a consistent process for preparing H&E slides from FFPE tissue samples using the automated H&E Leica Stainer and coverslip instruments. This procedure is performed by trained histology laboratory personnel.

Proper citation: Linda Thomas, Angie Brown, Kerry Wiles 2021. Automated H&E Staining and Coverslipping (Leica). protocols.io https://dx.doi.org/10.17504/protocols.io.bpyhmpt6 Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Phenotyping of Rodents for the Presence of Diabetic Neuropathy In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.Diabetic Complication:

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Cryoembedding. protocols.io https://dx.doi.org/10.17504/protocols.io.3jpgkmn Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare 10% Tween 20. Tween 20, when added to aqueous solutions acts as a surfactant, decreasing the surface tension of the solution. It also punches holes in membranes, allowing easier penetration of macromolecules into cells and organelles, and reduces non-specific binding of antisera in immunohistochemistry.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. 10% Tween 20. protocols.io https://dx.doi.org/10.17504/protocols.io.bfzmjp46 Copy   

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Authors: Roland Wouters, Sam Mugford, Roberto Biello, Darren Heavens, Saskia Hogenhout
Group: High molecular weight DNA extraction from all kingdoms
Summary: Chromosome-scale genome assembly usually requires the use of long-read sequencing technologies such as PacBio or ONT MinION. However, the development of suitable DNA extraction methods that can yield high molecular weight DNA suitable these platforms can be problematic and time-consuming. We found that some traditional DNA extraction methods that are suitable for short-read sequencing platforms did not produce satisfactory results using long-read sequencing technologies with aphids or other sap-feeding hemipteran insects. We hypothesised that plant-derived components of the aphid’s diet might inhibit the sequencing chemistry. Here we present a method suitable for extraction of high-molecular DNA for long-read sequencing of aphids and other sap-feeding insects. We employed the Illustra Nucleon Phytopure DNA extraction kit designed to remove plant-specific contaminants. We were able to consistently recover very high molecular weight DNA that was compatible with library preparation and sequencing by PacBio or ONT MinION. The protocol presented here describes the use of this plant-DNA extraction kit optimised to obtain high molecular weight DNA from insect samples.We have used this method across a range of aphid species, leafhoppers and froghoppers (spittlebugs). It has enabled sequencing and - in most cases -chromosome-level assembly of genomes from the green peach aphid Myzus persicae (1), the pea aphid Acyrthosiphon pisum (1), the woolly apple aphid, Eriosoma lanigerum (2), and the common meadow spittlebug Philaenus spumarius (3). 1. Chromosome-scale genome assemblies of aphids reveal extensively rearranged autosomes and long-term conservation of the X chromosomeThomas C.Mathers, Roland H. M.Wouters, Sam T.Mugford, DavidSwarbreck, CockVan Oosterhout, Saskia A.HogenhoutbioRxiv 2020.03.24.006411; doi: https://doi.org/10.1101/2020.03.24.006411 2. A chromosome-level genome assembly of the woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae)RobertoBiello, ArchanaSingh, Cindayniah J.Godfrey, FelicidadFernández Fernández, Sam T.Mugford, GlenPowell, Saskia A.Hogenhout, Thomas C.MathersbioRxiv 2020.05.29.121947; doi: https://doi.org/10.1101/2020.05.29.121947 3. Draft genome assembly version 1 of the meadow spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) (Version 1) [Data set]. Roberto Biello, Thomas C. Mathers, Sam T. Mugford, Qun Liu, Ana S. B. Rodrigues, Ana Carina Neto, Maria Teresa Rebelo; Octávio S. Paulo; Sofia G. Seabra; Saskia A. Hogenhout. (2020). Zenodo. http://doi.org/10.5281/zenodo.3368385

Proper citation: Roland Wouters, Sam Mugford, Roberto Biello, Darren Heavens, Saskia Hogenhout 2020. Extraction of high molecular weight DNA from aphids and other sap-feeding insects for long-read sequencing.. protocols.io https://dx.doi.org/10.17504/protocols.io.bhftj3nn Copy   

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Authors: Doug Campbell, Natalie Donaher
Summary: The Campbell Lab protocol for imaging Western Blots using ECL Select chemi-luminescent detection and the Bio-Rad VersaDoc imager for immunoquantitation of phytoplankton samples.

Proper citation: Doug Campbell, Natalie Donaher 2017. Image Western Blots for quantitative immunoblotting using the Bio-Rad VersaDoc CCD camera. protocols.io https://dx.doi.org/10.17504/protocols.io.jwscpee Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for how to perform DNA ligation. To see the full abstract and other resources, visit https://www.addgene.org/protocols/dna-ligation/.

Proper citation: Addgene The Nonprofit Plasmid Repository 2019. DNA Ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.4g7gtzn Copy   

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Authors: Demian F Gomez, Andrew J. Johnson, You Li
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how to run electrophoresis gels at the UF Forest Entomology Lab.This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Demian F Gomez, Andrew J. Johnson, You Li 2020. Running Gels: Electrophoresis Gel Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.bnvdme26 Copy   

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