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Authors: Igem Dusseldorf

Proper citation: Igem Dusseldorf 2019. Glycerol stocks . protocols.io https://dx.doi.org/10.17504/protocols.io.8e3htgn Copy   

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Authors: Michael Frimpong, Hubert Senanu Ahor
Summary: This document describes the standard operating procedure for the application of the real time Mu-RPA assay with Exo probe system. The assay detects M. ulcerans DNA (IS2404) from clinical sample or culture suspension. The assay consists of recombinase polymerase amplification TwistDx Exo kit procedure. The reaction mix must be prepared in an environment free of DNA amplicons. Personal protective clothing (i.e. lab coats, gloves) must be used throughout the process.

Proper citation: Michael Frimpong, Hubert Senanu Ahor 2019. Recombinase polymerase amplification assay for detection of Mycobacterium ulcerans DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.vvve666 Copy   

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Authors: Juan Wu
Summary: In the current study, we successfully established an acute inflammation model and a chronic inflammation model involving adipose stem cells. We used high-throughput miRNA microarray analysis to identify miRNAs that were significantly (pwhich was directly targeted by miR-223-3P. STAT3 directly targeted TLR4 and TLR2, promoting the production of the pro-inflammatory cytokine, IL-6, and formed a positive feedback loop to regulate IL-6 levels. Similarly, TNF-a significantly(p1.Establish an acute inflammation model of adipose stem cells2.Establish a chronic inflammation model of adipose stem cells3. miRNA microarray and data analysis4.Target gene prediction, gene ontology (GO) enrichment, and pathway analysis5.Prediction of target gene binding sites6.Dual luciferase assay7.LPS induces STAT3mRNA and protein levels in adipose stem cells, and promotes STAT3 protein phosphorylation, while miR-223-3p regulates STAT3 mRNA8.miR-223-3p directly regulates TLR2 and TLR4, and the secretion of inflammatory cytokines IL-6, TNF-a, and IL-1b9. STAT3 promotes phosphorylation of STAT3 protein, and regulates TLR4 and TLR2 expression10.Treatment of adipose stem cells with IL-6, TNF-a, siIL-6, and siTNF-a affects the miR-223-3p, STAT3, and pSTAT3 levels11.miR-2909 depends on NF-kB to target KLF4 to regulate IL-6, IL-1b, and TNF-a levels

Proper citation: Juan Wu 2018. Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1, and TNF-, and the TLR4/TLR2/NF-B/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.vzhe736 Copy   

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Authors: Ian Mcloughlin
Summary: The task described here is that used in the PLoS ONE paper entitled "Continuous Robust Sound Event Classification Using Time-Frequency Features and Deep Learning". It builds upon the long established standard isolated sound evaluation task first described by Jonathan Dennis, and widely used by a number of other authors - for evaluating classifiers of isolated sounds.By contrast the current task extends this to potentially overlapping sounds with no a priori knowledge of start and end points. The advantage of having a standard task is obvious: it makes experiments easily repeatable by others, and eases the comparison of results when other authors make use of the same method to evaluate their own research. With at least 15 state-of-the-art sound classification papers published with Dennis' isolated sound event detection method, it is easily the most popular task defined to date.In this current task, exactly the same raw material is used, but is extended through protocol and setup into a continuous, overlapping and robust classification task. The task uses freely available sound recordings from the Real World Computing Partnership (RWCP) Sound Scene Database in Real Acoustic Environments. These must be obtained directly by the RWCP, and are free for non-commercial or academic users, while commercial users are charged a small free.Robustness evaluation is performed by mixing raw sounds with background noises from the NOISEX-92 database at several signal-to-noise (SNR) levels. The NOISEX-92 data is widely available online for download.

Proper citation: Ian Mcloughlin 2017. Continuous Robust Sound Event Classification Task. protocols.io https://dx.doi.org/10.17504/protocols.io.iw5cfg6 Copy   

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Authors: Daniel Gyllborg, Chika Yokota, Mats Nilsson
Group: Human Cell Atlas Method Development Community, Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: In situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved tissue. This protocol can be used to detect RNA molecules at the single cell level to aid in the identification of cell types according to their gene expression. The technique uses padlock probes to target desired genes of interest and rolling circle amplification to amplify signal for a high throughput methodolgy of spatial transcriptomics. With the use of barcode sequencing, identification of numerous genes is possible through multiplexing.

Proper citation: Daniel Gyllborg, Chika Yokota, Mats Nilsson 2019. In situ sequencing for RNA analysis in tissue sections. protocols.io https://dx.doi.org/10.17504/protocols.io.s8vehw6 Copy   

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Authors: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar
Group: SPARC
Summary: This protocol is used to surgically implant gastrointestinal planar electrodes and vagus nerve cuff electrodes for chronic electrophysiological recording and stimulaiton in the ferret. Electrode lead wiring is tunneled subcutaneously to a connector embedded in a headcap affixed to the cranium.Funding: This protocol was developed with funding from the NIH Common Fund’s Stimulating Peripheral Activity to Relieve Conditions (SPARC) program (Award U18TR002205). To learn more about the SPARC program, visit https://sparc.science.

Proper citation: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar 2019. SPARC - Chronic implantation of gastrointestinal and vagus nerve electrodes in the ferret. protocols.io https://dx.doi.org/10.17504/protocols.io.6crhav6 Copy   

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Authors: Jesica Palacio, Lily Johanna Toro, Germán Alberto Téllez Ramírez, Juan Pablo Bedoya Agudelo, Diana Carolina Henao, Jhon Carlos Castaño Osorio
Group: Grupo de inmunología molecular
Summary: El ensayo de herida o ruptura in vitro, es un método fácil, de bajo costo y bien desarrollado para medir migración celular in vitro. Los pasos básicos implican la creación de una “herida” en una monocapa celular, capturando las imágenes al principio  y en intervalos regulares de tiempo durante la migración celular para cerrar la herida y comparar las imágenes para cuantificar la tasa migración de las células. La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012. 

Proper citation: Jesica Palacio, Lily Johanna Toro, Germán Alberto Téllez Ramírez, Juan Pablo Bedoya Agudelo, Diana Carolina Henao, Jhon Carlos Castaño Osorio 2017. ENSAYO DE CICATRIZACIÓN IN VITRO, PARA EVALUAR MIGRACIÓN CELULAR. protocols.io https://dx.doi.org/10.17504/protocols.io.kdics4e Copy   

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Authors:
Group: UCSC BME 22L

Proper citation: 2020. Protocols for CRISPR. protocols.io https:// Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR for Parapoxvirus targeting the DNA polymerase. It is used to screen human samples where Parapoxvirus is suspected.This protocol is based on the published RVSS assay by Das et al 2017. Oligonucleotides have been modified and a different PCR kit is used.

Proper citation: Judy Northill, Ian Mackay 2018. Parapoxvirus real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.p4kdquw Copy   

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Authors: Song Chen, Blue B. Lake, Sarah Urata, Kun Zhang
Group: Human Cell Atlas Method Development Community, KPMP
Summary: The protocol presented here is a Drop-Seq protocol modified for single nuclei. The original Drop-Seq protocol comes from the McCarroll Lab in the Department of Genetics, Harvard Medical School.http://mccarrolllab.org/download/905/

Proper citation: Song Chen, Blue B. Lake, Sarah Urata, Kun Zhang 2020. Single Nucleus Drop-seq (snDrop-seq). protocols.io https://dx.doi.org/10.17504/protocols.io.zmvf466 Copy   

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Authors: Cj Xia
Group: Boster Bio
Summary: Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different subpopulations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell. The number of measurable parameters that can be used by this technology to separate cell populations is immense – starting from simple surface immunophenotyping to metabolic functions, cell cycle status, redox state, and DNA content analysis to name a few. Since its inception, FACS has been used extensively in biomedical research and clinical diagnostics and therapeutics. The most common usage of FACS is seen in: - Analysis of whole human blood for diagnosing diseases, immunophenotyping - Sorting different blood cell fractions for ex-vivo manipulations and/or transplantations - Immuno-phenotypic analysis of murine blood to identify transgenic/knockout animals - Sorting and analysis of a slew of cell lines for various biological assays - Characterization and isolation of rare cells types like adult stem cells and cancer initiating cells Each human cell expresses hundreds of thousands of cell surface antigens that specify their cell type, biological function, development stage, and much more. Cells residing in different organs have characteristic cell surface antigens, and determination of these cells using the specific fluorophore-conjugated antibodies can be analyzed by flow cytometry. The following general protocols are recommended for various common FACS staining procedures. Staining with unconjugated purified antibody needs an additional step of staining with a fluorescent conjugated secondary antibody (indirect immunostaining).

Proper citation: Cj Xia 2018. Flow Cytometry Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mgdc3s6 Copy   

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Authors: Diogo Provete

Proper citation: Diogo Provete 2016. Fixing solution to SEM. protocols.io https://dx.doi.org/10.17504/protocols.io.e3zbgp6 Copy   

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Authors: Joshua Timmons
Group: Northeastern iGEM 2015
Summary: Northeastern's method for plasmid extraction and purification. From Thermo Scientific's GeneJET Plasmid Miniprep Kit

Proper citation: Joshua Timmons 2015. Plasmid Miniprep. protocols.io https://dx.doi.org/10.17504/protocols.io.dp95r5 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Streptozotocin can selectively destroy the pancreatic β-cells with rapid and irreversible necrosis and can be used to generate a chronic model of hyperglycemia and type 1 diabetes.

Proper citation: Jason Kim 2019. U Mass - STZ-induced type 1 diabetes model. protocols.io https://dx.doi.org/10.17504/protocols.io.xh8fj9w Copy   

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Authors: Tory Stoddard, Quint Heyder, Alyssa Leonardi, Andrew Saltzman
Summary: Fish egg disinfection is a common practice in aquaculture facilities to prevent disease and improve the hatch rates of fish eggs. This process helps remove bacteria, fungus, and other unwanted organisms from eggs. Iodine was found to be the most effective disinfecting agent based on available literature and recent experimentation. 

Proper citation: Tory Stoddard, Quint Heyder, Alyssa Leonardi, Andrew Saltzman 2019. A Comparison of the Performance of Disinfection Agents on Smallmouth Grunt (Haemulon chrysargyreum) Eggs. protocols.io https://dx.doi.org/10.17504/protocols.io.baqxidxn Copy   

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Authors: Angela Piersanti, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Euplotes crassus GFP-neo artificial nanochromosomes

Proper citation: Angela Piersanti, Rachele Cesaroni 2019. Euplotes crassus GFP-neo artificial nanochromosomes sequence. protocols.io https://dx.doi.org/10.17504/protocols.io.2atgaen Copy   

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Authors: Afiahayati, Sato K, Namiki T, Hachiya T, Tanaka H, Sakakibara Y.
Group: VERVE Net, Hurwitz Lab
Summary: Bambus2 is a scaffolding module that can be applicable to metagenomics settings. MetaVelvet uses a novel graph splitting algorithm during contiging process, and uses the scaffolding module of Velvet (RockBand and Pebble) during scaffolding process. Alternatively, users can output MetaVelvet contigs and uses Bambus2 scaffolding module instead of using RockBand and Pebble.

Proper citation: Afiahayati, Sato K, Namiki T, Hachiya T, Tanaka H, Sakakibara Y. 2016. Use Bambus2 scaffolding module. protocols.io https://dx.doi.org/10.17504/protocols.io.d7n9md Copy   

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Authors: Andrew Crowley
Group: Ackerman Lab
Summary: A protocol for the preservation of suspension-type cells by freezing.The protocol has been sucessfully used on: - THP-1 monocytes

Proper citation: Andrew Crowley 2019. Cryopreservation of Mammalian Cells (Suspension). protocols.io https://dx.doi.org/10.17504/protocols.io.47pgzmn Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol outlines strategies to solve common problems that occur when setting up the TissueCyte 1000 system for normal operation.

Proper citation: Allen Institute for Brain Science 2020. TissueCyte Troubleshooting Guide. protocols.io https://dx.doi.org/10.17504/protocols.io.bbyaipse Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the basic setup and scanning using the Leica SP8 confocal microscope of fluorescently-labeled mouse brain tissue sections mounted on positively charged 1” x 3” microscope slides.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. Leica SP8 Confocal Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.bd8wi9xe Copy   

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