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Authors: Cellular Generation and Phenotyping
Group: Cellular Generation and Phenotyping
Summary: This protocol outlines the method for thawing, passaging and cryopreserving established feeder-free induced pluripotent stem cell lines. In this culture method, cells are kept as aggregates throughout the process. Cells are cultured on vitronectin matrix (full length xeno-free or truncated protein) in E8 or TeSR-E8 media. Cells can be cultured with or without Penicillin-Streptomycin in the media. Cell lines cultured using these protocols are suitable to use for downstream applications including differentiation and CRISPR screens.

Proper citation: Cellular Generation and Phenotyping 2020. Culture of established induced pluripotent stem cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bgbwjspe Copy   

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Authors: Olabode Omotoso
Group: Cancer Research and Molecular Biology Laboratories, University of Ibadan
Summary: This protocol outlines the methodology for the acquisition and analysis of SARS-CoV-2 genomic sequences.

Proper citation: Olabode Omotoso 2020. SARS-CoV-2 Genomic Variation - African perspective. protocols.io https://dx.doi.org/10.17504/protocols.io.bmpfk5jn Copy   

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Authors: Keizo Nagasaki and Gunnar Bratbak
Group: VERVE Net
Summary: This is a detailed outline of the extinction dilution method from: Nagasaki, K., and G. Bratbak. 2010. Isolation of viruses infecting photosynthetic and nonphotosynthetic protists, p. 92–101. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.Please see the published manuscript for additional information.

Proper citation: Keizo Nagasaki and Gunnar Bratbak 2015. Extinction dilution cloning for isolation of viruses infecting protists. protocols.io https://dx.doi.org/10.17504/protocols.io.dpx5pm Copy   

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Authors: Marmar Vaseghi, Jeffrey Ardell

Proper citation: Marmar Vaseghi, Jeffrey Ardell 2019. Pig Nodose Ganglion protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.2ncgdaw Copy   

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Authors: LeinU01 BRAIN Grant
Group: BICCN, Allen Institute for Brain Science
Summary: Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for FPCR and/or RNA-seq analysis.

Proper citation: LeinU01 BRAIN Grant 2019. Isolation of Nuclei from Adult_Human_Brain_Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.ztqf6mw Copy   

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Authors: Mark Wilkinson, Mario Prieto Godoy
Summary: Using the TAC Biomedical Summarization Corpus (Min-Yen), we extracted 45 manually­ curated scholarly assertions (selection described below). Based on these, we generated three independently-executed Web­-based questionnaires where researchers in the biomedical domain, from different linguistic groups but in comparable research institutes, were presented with assertions and asked to categorize the strength of those assertion into 4 (High, Medium High, Medium Low and Low), 3 (Category 1, Category 2 and Category 3) or 2 (Relatively High and Relatively Low) certainty levels over the three questionnaires. G Index (Holley & Guilford, 1964) coefficient analysis was applied to determine the degree of agreement between annotators. We then extracted the essential features of inter-rater agreement from the questionnaire data using Principal Component Analysis (PCA). Afterward, we categorized our collection of statements in clusters using k-means algorithm (Jolliffe, 2011) (Dunham, 2006). Finally, an automated classifier model was generated using deep-learning techniques over the results of this study (manuscript under preparation). This was used to construct exemplar scholarly assertions capturing certainty metadata, and published as machine-readable NanoPublication.

Proper citation: Mark Wilkinson, Mario Prieto Godoy 2019. Scholarly Certainty Survey and Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.x6nfrde Copy   

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Authors: Chantal Vogels, Joseph Fauver, Nathan Grubaugh
Group: Coronavirus Method Development Community
Summary: As the B.1.1.7 "UK" variant continues to spread, there is a need for labs to screen COVID-19 samples for these viruses. While sequencing is the gold standard, it cannot always be scaled or implemented in some settings. The Applied Biosystems TaqPath COVID-19 assay (ThermoFisher), a PCR test, was discovered to have a distinct signature (spike gene target failure, [SGTF]) when testing viruses containing the Δ69/70 HV deletion, like the B.1.1.7 variant. Detecting the Δ69/70 HV deletion alone is not definitive for the B.1.1.7 variant, but tracking the frequencies of SGTFs helped the UK track the B.1.1.7 variant. In the US and other countries, screening samples for the SGTF helped to identify potential B.1.1.7 variants for sequencing prioritization. ThermoFisher, however, has not released their spike probe sequence, so the assay needs to be recreated to be used more broadly. Here, we designed a multiplexed PCR assay that recreates the SGTF signature using primer/probe set that targets the Δ69/70 HV deletion, the CDC N1 set as a positive control, and the CDC RNase P set as an extraction/sample control. Samples positive for the CDC N1 set but negative for the Δ69/70 HV deletion set can be tracked and prioritized for sequencing.

Proper citation: Chantal Vogels, Joseph Fauver, Nathan Grubaugh 2021. Multiplexed RT-qPCR to screen for SARS-COV-2 B.1.1.7 variants. protocols.io https://dx.doi.org/10.17504/protocols.io.brcrm2v6 Copy   

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Authors: Alexander Ordynets, Sarah Keßler, Christina Willemsens
Summary: The protocol explains extracting and quantifying the shape and size information from the fungal spores. The complex information about the outline will be transformed into few values that can be used as shape descriptors in comparative analyses. The photos of spores from the light microscopy serve as input data. The shape information is processed with the elliptic Fourier analysis and afterward with the principal component analysis (PCA). The size information is captured as linear measurements of length and width. The average per specimen values for shape and size traits are then calculated in R computing environment. The protocol can be also applied to the images from other kinds of microscopy or to images of macroscopic objects (plants, animals, or their organs). The protocol contains example files and screenshots that should assist in the processing of own data. See "Materials" for the list of used software and "Guidelines and Warnings" for the flowchart illustrating the protocol.

Proper citation: Alexander Ordynets, Sarah Keßler, Christina Willemsens 2021. Extracting shape and size information from fungal spores. protocols.io https://dx.doi.org/10.17504/protocols.io.bdeii3ce Copy   

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Authors: Wen Aw
Group: Cage Studies

Proper citation: Wen Aw 2018. Metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.rtad6ie Copy   

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Authors: Nick Irwin, Elisabeth Hehenberger, Liz Cooney, Yoshihisa Hirakawa, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: INTRODUCTIONHere we describe a protocol for transforming Oxyrrhis marina with FITC-labelled dextran (a DNA analog) by electroporation. This protocol is reproducible, and results in a transformation efficiency of around 1%.We have confirmed the viability of transformed cells by allowing them to recover in the dark (and thus maintain fluorescence intensity) and then by observing fluorescent cells swimming normally, days after transformation (see the video here). NOTE ON AUTOFLUORESCENCEAlthough Oxyrrhis is a non-photosynthetic dinoflagellate and therefore lacks plastid autofluorescence, we have observed a small amount of green autofluorescence that is typically isolated in the anterior of the cell (See top panel of Figure 1).The autofluorescence was observed in wildtype cells, cells electroporated without FITC-dextran, and cells that were incubated with FITC-dextran but not electroporated. The autofluorescence was observed in cultures with both Dunaliella tertiolecta or heat-killed E. coli. The brightness and frequency of the autofluorescence also seemed to be dependent on culture conditions. Not all cells had the autofluorescence.ADDITIONAL INFORMATIONAs of yet we have been unable to demonstrate the actual uptake of plasmid DNA. We have tried using rhodamine labelled plasmids to demonstrate this but the results were inconclusive due to issues with cellular autofluorescence. We have also tried this method with a variety of expression plasmids encoding different promoters. For example, we tried the MOE promoter from Perkinsus marinus (a closely related species) and the generic eukaryotic CMV promoter. However no GFP fluorescence was detected in these samples. We have also tried transforming in vitro synthesized GFP encoding mRNA using this protocol. As of yet, no GFP production has been detected, but this may reflect non-cannonical translational systems in dinoflagellates as the same mRNA could be translated in starfish embryos.Although the development of expression systems in dinoflagellates remains a challenge, our results suggest that O. marina may be amenable to genetic transformation.

Proper citation: Nick Irwin, Elisabeth Hehenberger, Liz Cooney, Yoshihisa Hirakawa, Patrick Keeling 2019. Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate.. protocols.io https://dx.doi.org/10.17504/protocols.io.3cmgiu6 Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2017. Direct-Blot™ Western Blotting Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.hvzb676 Copy   

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Authors: Savi Eleonora, Peveri Silvia, Montagni Marcello

Proper citation: Savi Eleonora, Peveri Silvia, Montagni Marcello 2017. CAP inhibition in hymenoptera venom allergy. protocols.io https://dx.doi.org/10.17504/protocols.io.ht7b6rn Copy   

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Authors: Noreen Wauford

Proper citation: Noreen Wauford 2016. PCR of mouse PCSK9 from cDNA (cleavage template). protocols.io https://dx.doi.org/10.17504/protocols.io.e87bhzn Copy   

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Authors: Sara Da Silva Nascimento, Pierre Teodosio
Summary: Biosensors are small devices that use biological reactions to detect target analytes. Such devices combine a biological component with a physical transducer, which converts bio-recognition processes into measurable signals. Its use brings a number of advantages, as they are highly sensitive and selective, relatively easy in terms of development, as well as accessible and ready to use. Biosensors can be of direct detection, using a non-catalytic ligand, such as cell receptors and antibodies, or indirect detection, in which there is the use of fluorescently marked antibodies or catalytic elements, such as enzymes. They also appear as bio-affinity devices, depending only on the selective binding of the target analyte to the ligative attached to the surface (e.g., oligonucleotide probe).

Proper citation: Sara Da Silva Nascimento, Pierre Teodosio 2020. Protocol: Phylogenetics analysis of TP53 gene in humans and its use in biosensors for breast cancer diagnosis. protocols.io https://dx.doi.org/10.17504/protocols.io.biz9kf96 Copy   

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Authors: G-Biosciences
Group: G-Biosciences

Proper citation: G-Biosciences 2016. CB-X™ Protein Assay for simple aqueous samples. protocols.io https://dx.doi.org/10.17504/protocols.io.e33bgqn Copy   

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Authors: Dr. Steven Wilhelm
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 2017. Plaque Assay For Screening Viral Concentrates for Bacteriophage. protocols.io https://dx.doi.org/10.17504/protocols.io.igxcbxn Copy   

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Authors: Masatsugu Okamura, Yuki Kataoka, Shunsuke Taito, Takashi Fujiwara, Atsushi Ide, Hideyuki Oritsu, Masashi Shimizu, Yoshitaka Shimizu, Ryoko Someya, Masaaki Konishi
Summary: Background Early mobilization for inpatient acute heart failure has been applied in clinical practice, but there is no consensus on the specific timing, efficacy, and safety of its initiation. For patients with heart failure, exercise therapy when their condition has stabilized has been shown to improve quality of life and reduce readmission rates. Although scoping reviews have been reported on early mobilization after acute myocardial infarction, no systematic review of early mobilization in patients with heart failure who are older and have a higher rate of multiple disabilities and physical deterioration than those with myocardial infarction has been reported, and its definition, efficacy, and safety have not been fully clarified. In heart failure, physical functions such as grip strength and activity of daily living (ADL) have been found to be associated with distant outcomes such as readmission rates and mortality, and early mobilization for acute heart failure, initiated to prevent physical conditioning and decline in ADLs, can contribute to these outcomes.Purpose The purpose of this study is to conduct a scoping review and systematic review of early mobilization for hospitalized heart failure to clarify its current definition and to evaluate its effectiveness and safety.Method and analysis[Part 1]Scoping review of early mobilization for heart failure (clinical definition)The purpose of our scoping review is to clarify current clinical definition of early mobilization for acute heart failure.We describe this protocol that follows the PRISMA extension for scoping reviews (PRISMA-ScR) statement. We also use the scoping review framework by The Joanna Briggs Institute (JBI) following five stages approach: (1) identifying the research question; (2) identifying relevant studies; (3) study selection; (4) charting the data; and (5) collating, summarizing, and reporting the results.[Part 2]Systematic review and meta-analysis of early mobilization for acute heart failureBased on the scoping review above, we will design PICO and conduct a systematic review and meta-analysis to examine the efficacy and safety of early mobilization for acute heart failure.We used a systematic review protocol template (dx.doi.org/10.17504/protocols.io.biqrkdv6). We followed the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 for preparing this protocol.Ethics and dissemination In this systematic review, only data from previously conducted studies will be analyzed. We will publish this systematic review in peer-reviewed journals.

Proper citation: Masatsugu Okamura, Yuki Kataoka, Shunsuke Taito, Takashi Fujiwara, Atsushi Ide, Hideyuki Oritsu, Masashi Shimizu, Yoshitaka Shimizu, Ryoko Someya, Masaaki Konishi 2021. Early mobilization for acute heart failure: a systematic review and meta-analysis protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.btzynp7w Copy   

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Authors: Anna Maria Tsakiroglou, Catharine West, Sue Astley, Kim Linton, Martin Fergie, Richard Byers
Summary: Observing immune cell infiltration and spatial organisaion in the tumour microenvironment of follicular lymphoma is important for the development of novel biomarkers for prognosis and treatment selection. However the identification of multiple immune cell sub-populations is limited in routine single-plex immunohistochemical staining. Our goal was to develop and validate an automated protocol for multiplex immunofluoresence using tyramide signal amplification, the Opal™ 7 color Kit (Akoya Biosciences) and the Ultra Discovery (Roche) autostainer, which would enable concurrent visualisation on the same tissue sections of:T follicular helper cells (CD4+)cytotoxic T cells (CD8+)Tregs (CD4+FOXP3+)Macrophages (CD68+)PD1+ lymphocytesDAPI nuclear counterstainWe aimed in addition to observe the spatial arrangement of these cells in reference to the follicle areas, therefore the CD21 marker, which is expressed in mature B cells and follicular dendtritic cells located in the follicles was added in the panel. We validated quantitatively the agreement between single-plex and multiplex assays by comparing stained area in sequential sections of a follicular lymphoma FFPE tissue micro-array (TMA). The TMA was constructed from a follicular lymphoma cohort of 44 patients collected retrospectively from the archives of the Christie NHS Foundation Trust (Manchester), with ethical permission granted by the Central Manchester Multi-centre Research Ethical Committee (03/08/016). The protocol was automated with the DISCOVERY ULTRA IHC/ISH Roche autostainer and stained areas were quantified using the Indica Labs’ Area Quantification Fluorescence module (HALO software).Statistically significant linear correlations were seen between single-plex and multiplex assays (R² >0.72, p-14, see attached) for all markers.Therefore, this multiplex immunofluorescence (mIF) protocol can be used to conserve tissue and observe immune cell spatial organisation in follicular lymphoma.

Proper citation: Anna Maria Tsakiroglou, Catharine West, Sue Astley, Kim Linton, Martin Fergie, Richard Byers 2019. Automated Multi-plex Immunofluorescence with TSA for CD4, CD8, FOXP3, CD21, PD1 and CD68 in Follicular Lymphoma. protocols.io https://dx.doi.org/10.17504/protocols.io.49ygz7w Copy   

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Authors: Mariana Rius, Joshua Rest
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: The purpose of this protocol is to evaluate the significance of delta G values, as a strategy to determine the significance of secondary structure. Sequences are shuffled randomly 100x, maintaining base composition but not base order. The percentile of the delta G vlaue of the non-scrambled (original) sequence is obtained from the distribution created by the delta G values of the 100X scrambled sequences.

Proper citation: Mariana Rius, Joshua Rest 2017. Genome-wide Kozak Sequence Free Energy Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.h9mb946 Copy   

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Authors: Koen Vandelannoote
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Koen Vandelannoote
Summary: Here, we describe a semi-automated workflow for paramagnetic bead-based extraction of SARS-CoV-2 RNA from human upper respiratory samples that uses low-cost repurposed Creality3D Ender-3 3D-printers and readily accessible reagents. The protocol described here is for the use of four instruments in parallel, each processing 24 samples, 96 samples per batch.

Proper citation: Koen Vandelannoote 2020. High-throughput SARS-CoV-2 RNA extraction from nasopharyngeal swabs using repurposed 3D printers.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkynkxve Copy   

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