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Authors: Michael Cheng, Xue-Zhong Ma, Chao Jiang, Ian McGilvray, Sonya Macparland
Summary: Analyzing the mouse liver requires optimal cell recovery from the tissue in terms of quality (viability) and quantity. Harsh dissociation methods can damage fragile cell populations, resulting in low viability and difficulty in cell culture. We have developed a gentle surgical and enzymatic technique based on the two-step collagenase perfusion protocol for single-cell analysis and primary cell culture.
Proper citation: Michael Cheng, Xue-Zhong Ma, Chao Jiang, Ian McGilvray, Sonya Macparland 2020. Mouse 2- Step Collagenase Liver Perfusion protocol . protocols.io dx.doi.org/10.17504/protocols.io.53qg8mw Copy
Authors: Daniel Marchal
Proper citation: Daniel Marchal 2018. Preparation of glycerol stocks for V. natriegens. protocols.io dx.doi.org/10.17504/protocols.io.uy2exye Copy
Authors: Gage Moreno, David O'connor
Group: Coronavirus Method Development Community
Summary: This protocol outlines the methods to perform unbiased direct metagenomic sequencing of nucleic acid extracts from cell-free fluids. This protocol can be adapted to be run on Illumina and Nanopore sequencing platforms. The protocol is based off of the work from Kafetzopoulou et al. (PMID: 30563591). Liana has provided the lab with detailed protocols, and has worked with us extensively on optimizing and getting protocols running efficiently. Please note that this protocol has been updated to use SuperScript IV with it’s optimal temperature which has been reflected in Lewandowski et al. (DOI: https://doi.org/10.1128/JCM.00963-19)Notes:* This protocol has been used to sequence influenza direction from respiratory clinical samples (DOI: https://doi.org/10.1128/JCM.00963-19).* A team from China published last week on a 2019-nCoV familial cluster using the SISPA protocol as for coronavirus whole genome sequencing (DOI: https://doi.org/10.1016/S0140-6736(20)30154-9)SISPA-Primer A - 5'-GTT TCC CAC TGG AGG ATA-(N9)-3'SISPA-Primer B - 5′-GTT TCC CAC TGG AGG ATA-3′
Proper citation: Gage Moreno, David O'connor 2020. Sequence-Independent, Single-Primer Amplification of RNA viruses. protocols.io dx.doi.org/10.17504/protocols.io.bhk4j4yw Copy
Authors: Sven Reischauer
Summary: Zebrafish are an increasingly popular model system to better understand vertebrate physiology. Physical challenge by exercise has become popular to assess physical fitness of genetically manipulated animals or to identify gene regulatory networks that prepare the organism for higher physical demands. The increasing popularity of the zebrafish model system to address questions surrounding physical fitness and physiology has led to the development and commercial availability of sophisticated exercise setups (e.g. swim tunnel respirometers, Loligo® Systems, Viborg, Denmark). However, in many cases, these commercial systems are not available to researchers as they are costly.This protocol describes a simple and effective way to exercise zebrafish. The setup is assembled from components that are present in virtually any laboratory. Its adoptable to constant or increasing endurance exercise as well as interval exercise or sprint tasks. The procedure does not cause stress to the animals as it relies on their natural behavior to swim against water currents to hold their position relative to their visual environment.
Proper citation: Sven Reischauer 2018. Assessing vertebrate physiology: A simple and cost-effective method to exercise adult zebrafish.. protocols.io dx.doi.org/10.17504/protocols.io.sa2eage Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for in vitro transcription (IVT) reaction generating DIG-labeled anti-sense (AS) RNA from polymerase chain reaction (PCR) product for in situ hybridization (ISH), as well as fluorescent quantification of products using Ribogreen immediately following. This protocol is written for 96 reactions but can also be used for single reactions or in multiples of 96 by adjusting the amount of materials and equipment appropriately.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. Digoxigenin Labeled In Vitro Transcription and Ribogreen Quantification. protocols.io dx.doi.org/10.17504/protocols.io.bg4vjyw6 Copy
Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. HBSS/HEPES for ROS measurement in PMN. protocols.io dx.doi.org/10.17504/protocols.io.bhdcj22w Copy
Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in CviRI Purification From XZ-6E Virus Infected NC64A Chlorella.
Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. CviRI Buffer A. protocols.io dx.doi.org/10.17504/protocols.io.eswbefe Copy
Authors: Barbra Ferrell
Group: VERVE Net, Wommack/Polson Viral Ecology and Informatics Resources (VEIL) Lab
Summary: Viral Informatics Resource for Metagenome Exploration (VIROME) is a bioinformatics pipeline that classifies viral metagenome sequences after searching against annotated reference sequence databases (i.e., UniRef, SEED, ACLAME, COG, GO, KEGG, and PhageSEED) and a custom environmental database, Metagenomes Online (MgOl). VIROME’s compare tool allows users to compare any number of metagenomes to one another by generating a Biological Observation Matrix (biom) file that contain counts of occurrences of custom observations (comparison metrics), and a mapping text file that provides per-sample metadata (library IDs and descriptions). Quantitative Insights Into Microbial Ecology (QIIME) is an open-source software pipeline that allows users to interpret raw sequencing data and create graphical displays to interact with the data. QIIME is modular, providing users with the flexibility to select and integrate various applications. While QIIME has many capabilities and online tutorials, this SOP is limited to the steps a user will take to use VIROME’s compare tool output (biom and mapping files) to explore composition (what is in a sample), alpha-diversity (diversity within a sample), and beta-diversity (comparison of diversity between samples). This SOP generally follows guidance in online tutorials but with modifications unique to the VIROME Comparinator output analysis.
Proper citation: Barbra Ferrell 2016. QIIME: VIROME comparinator tool diversity metrics. protocols.io dx.doi.org/10.17504/protocols.io.ec9baz6 Copy
Authors: Benito Recio Totoro
Summary: Plasmodium berghei is one of the main non-human malaria models used in research. It readily infects laboratory strains of mice and rats as well as mosquitoes. Because of this, it is safe to manipulate and it does not require human fluids/tissues. This document provides the Operational Procedure for producing and culturing ookinetes of P. berghei in vitro. The method has been optimized for the P. berghei ANKA train clone 2.34 and the derived GFP-expressing clone, but should work for other strains.This method is adapted and modified from:
Proper citation: Benito Recio Totoro 2020. Plasmodium berghei ookinete culture. protocols.io dx.doi.org/10.17504/protocols.io.bmm8k49w Copy
Authors: Renuka Kudva
Summary: This protocol describes the procedure make an S30 derived E.coli cell lysate that lacks membrane components. Modified from a protocol published in https://doi.org/10.1091/mbc.e11-07-0590The procedure takes three days in total, including preparation time.
Proper citation: Renuka Kudva 2019. Preparation of a cell-free expression system from Escherichia coli. protocols.io dx.doi.org/10.17504/protocols.io.2xkgfkw Copy
Authors: Xiao-bei Si, Shuai Wang, Xu-min Zhang, Yu Lan
Summary: Background: In recent years, many studies reported the anti-Hp effects of Traditional Chinese Medicine. Based on these studies, there had been many relevant systemic reviews and meta analyses with variable resultsAim: This overview aims to investigate the general characteristics and assess the methodological quality of these systemic reviews/meta ana lyses (SR/MAs).Methods: We included systemic reviews/meta analyses with comparison of Chinese medicine and western medicine on helicobacter pylori infection by searching databases of Medline, Embase, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese VIP Information (VIP), Chinese Medical Databases (CMB) and Wan-Fang Database until December 16th, 2018. AMSTAR and PRISMA tools were adopted to assess the methodological quality and reporting characteristics respectively.
Proper citation: Xiao-bei Si, Shuai Wang, Xu-min Zhang, Yu Lan 2019. Traditional Chinese Medicine for Helicobacter Pylori infection: An Evidence Assessment for Systemic Reviews and Meta-analysesl. protocols.io dx.doi.org/10.17504/protocols.io.w28fghw Copy
Authors: G Jason Smith, April Woods
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: Use for solid phase culture and chemical selection of Pseudo-nitzschia spp and other recalcitrant diatoms
Proper citation: G Jason Smith, April Woods 2016. Low Gel Temperature Agarose (LGTA) Media. protocols.io dx.doi.org/10.17504/protocols.io.gmnbu5e Copy
Authors: Alise Ponsero
Group: iMicrobe, Metafunc course 2018
Summary: How to run Prodigal version 2.6.3 (Hyatt et al. 2010) through the iMicrobe plaform.Prodigal is a protein-coding gene prediction software tool for bacterial and archaeal genomes. Prodigal runs smoothly on finished genomes, draft genomes, and metagenomes. Please note that Prodigal has not been tested on viruses, and that Prodigal contains no special rules or routines to handle viral genomes.More informations about Prodigal can be found here : https://github.com/hyattpd/Prodigal/wiki
Proper citation: Alise Ponsero 2018. Run Prodigal using iMicrobe. protocols.io dx.doi.org/10.17504/protocols.io.s4gegtw Copy
Authors: Christopher Bartley
Summary: Protocol for using Mouse-FMRP antibody follow immunoprecipitation of FMRP using a rabbit antibody such as Abcam #17722
Proper citation: Christopher Bartley 2015. FMRP 1C3 Western Blot (Millipore MAB2160) post-IP. protocols.io dx.doi.org/10.17504/protocols.io.crev3d Copy
Authors: Kanae Nishii
Summary: R codes used for phyml tree and profile HMM heatmap visualization, protein domain illustration
Proper citation: Kanae Nishii 2021. R editing of HMMSEARCH output: heatmap with associated with gene tree and protein illustration. protocols.io dx.doi.org/10.17504/protocols.io.bkqwkvxe Copy
Authors: Valeria Jimenez
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff
Summary: This methodology describes a protocol modified from Sherr and Sherr (1993) to determine phytoplankton prey uptake using flow cytometry and yellow-green fluorescent polystyrene-based microspheres (YG-beads; 0.5 µm in diameter, Fluoresbrite, Polysciences, Inc., Warrington, PA, USA) or fluorescently labelled bacteria (FLBs) as food proxies. FLBs were prepared according to the protocol of Sherr et al. (1987) using the bacteria Brevundimonas diminuta (strain CECT313, also named Pseudomonas diminuta), obtained from the Spanish Type Culture Collection (CECT, Valencia, Spain). The percent of cells with prey were measured in the laboratory in four Micromonas polaris strains and one mixotrophic strain (Ochromonas triangulata) used as a positive control. The percent of cells with beads was determined in samples fixed with Lugol's iodine solution (protocol explained in detail here) and enumerated with a flow cytometer (Guava easyCyte, Luminex Corporation, USA) where cells that contained chlorophyll as well as green fluorescence (same signal as the prey added, YG-beads or FLBs) were considered to be cells containing prey (quantification protocol explain in detail here). To determine the uptake of prey over time the percent of cells with preys was quantified in each experimental flask by first adding preys and then sub-sampling and fixing after an incubation of 0 (T0), 20 (T20) and 40 (T40) minutes. The T0 sample accounts for the physical attachment of preys to the cell and therefore the percent of cells ingesting prey corresponds to the percent of cells with prey at T20 or T40, minus the percent of cells with prey at T0. References:Sherr, EB. & Sherr, BF. (1993). Protistan grazing rates via uptake of fluorescently labeled prey. In Kemp, P., Sherr, B., Sherr, E. & Cole, J. (eds.) Handbook of Methods in Aquatic Microbial Ecology, 695–701 (Lewis Publishers: Boca Raton, USA).Sherr, EB., Sherr, BF. & Fallon, RD. (1987). Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl Environ Microbiol.
Proper citation: Valeria Jimenez 2020. Mixotrophy - Quantification of the percent of phytoplankton cells with preys (e.g. fluorescent microspheres or fluorescently labeled bacteria). protocols.io dx.doi.org/10.17504/protocols.io.be2vjge6 Copy
Authors: Jacqueline Hogue, Natalie O. Rosen, Amanda Bockaj, Emily A. Impett, Amy Muise
Summary: In this project, we recruited a sample of couples coping with female sexual interest/arousal disorder (FSIAD) to investigate the role of sexual communal strength and unmitigated sexual communion in the sexual well-being and sexual goals of both women with FSIAD and their partners.
Proper citation: Jacqueline Hogue, Natalie O. Rosen, Amanda Bockaj, Emily A. Impett, Amy Muise 2019. Sexual communal motivation in couples coping with low sexual interest/arousal: Associations with sexual well-being and sexual goals. protocols.io dx.doi.org/10.17504/protocols.io.zn3f5gn Copy
Authors: Amparo Villablanca
Group: Mouse Metabolic Phenotyping Centers
Summary: Increased lipid accumulation in the artery wall and atherosclerotic lesion development are two hallmarks of atherosclerotic cardiovascular disease. Atheroma quantification and characterization assesses neutral lipid accumulation in the vessel wall, percentage of lesions per aortic segment (quantitative) and lesion morphology, complexity, and severity (qualitative).Modified from: Villablanca AC, et.al. J Cardiovasc Transl Res. 2009 Sep;2(3):289-99.
Proper citation: Amparo Villablanca 2019. UC Davis - Atheroma Quantification. protocols.io dx.doi.org/10.17504/protocols.io.7bwhipe Copy
Authors: Irina Agarkova
Group: VERVE Net, The Chlorovirus Group
Proper citation: Irina Agarkova 2016. Pulse Field Gel Electrophoresis (PFGE) Protocol for separation of Chlorella chromosomal DNA and Chlorella virus DNA. protocols.io dx.doi.org/10.17504/protocols.io.essbeee Copy
Authors: Gemma Curie
Group: Innovative Genomics Institute, CornLab
Proper citation: Gemma Curie 2015. T7 PCR Template for 1sgRNA Mixture. protocols.io dx.doi.org/10.17504/protocols.io.dna5ad Copy
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