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Authors: Jake Willows, Kristy Townsend, Magdalena Blaszkiewicz
Group: Townsend Lab Neurobiology & Energy Balance
Summary: AbstractLittle is known about the diversity and function of adipose tissue nerves due, in part, to the inability to effectively visualize the various nerve subtypes residing within these tissues. The tools currently available for researchers to image and quantify adipose tissue innervation are limited and dependent on optical clearing techniques and light sheet microscopy. Here we present a method of tissue processing that uses a method of mechanically compressing tissue to decrease tissue thickness in the z-axis by expanding it in the x and y-axes whilst leaving cells intact. This has been combined with autofluorescence quenching techniques to permit imaging of intact whole tissues on both widefield and confocal microscopes and a complementary means to perform whole tissue neurite density quantification. We have included examples of how this technique can be used to further our current knowledge of adipose-nerve communication by characterizing the nerves, nerve-subtypes, and neurovascular interactions within the inguinal subcutaneous white adipose tissue in mice.

Proper citation: Jake Willows, Kristy Townsend, Magdalena Blaszkiewicz 2021. Adipose depot innervation: whole mount staining, imaging, quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.brs2m6ge Copy   

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Authors: Rui Zhang
Group: BGI, GIGA, GigaScience Press
Summary: The humpback puffer,Tetraodon palembangensis, is a species of poisonous freshwater pufferfish mainly distributed in Southeast Asia (Thailand, Laos, Malaysia and Indonesia). The humpback puffer has many interesting biological features, such as inactivity, tetrodotoxin production and body expansion. Here, we reported the first chromosome-level genome assembly of the humpback puffer. The genome size is 362 Mb with ~1.78 Mb contig N50 and ~15.8 Mb scaffold N50. Based on the genome, ~61.5Mb (18.11%) repeat sequences were identified, 19,925 genes were annotated, and 90.01% of these genes could be predicted with function. Finally, a phylogenetic tree of ten teleost fish species was constructed, which suggests that humpback puffer and T. nigroviridis shared a common ancestor at 18.1 MYA and diverged from T. rubripes at 45.8 MYA. The humpback puffer genome will be a valuable genomic resource to illustrate possible mechanisms of tetrodotoxin synthesis and tolerance.

Proper citation: Rui Zhang 2021. Oxford Nanopore sequencing and library construction. protocols.io https://dx.doi.org/10.17504/protocols.io.btcwnixe Copy   

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Authors: Ms Alex Aitken
Group: VERVE Net, Sullivan Lab
Summary: TE is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation: 10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA

Proper citation: Ms Alex Aitken 2016. TE Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.c5uy6v Copy   

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Authors: Marta Tomaselli, Marius Rebmann
Group: OpenPlant Project
Summary: This protocol allows high throughput imaging of Marchantia gemmae, using a cheap setup made with broadly available lab equipment. We used a transparent 384 wells plate filled with 1/2 strength Gamborg B5 media with 1.2% agar and placed a single gemma at the centre of the well. Gene frames and coverslips treated with anti-fog spray were used to cover (seal) the wells. This setup can be paired with automated imaging of samples.

Proper citation: Marta Tomaselli, Marius Rebmann 2019. Marchantia high throughput imaging in multiwell plates. protocols.io https://dx.doi.org/10.17504/protocols.io.4v4gw8w Copy   

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Authors: Jing-Zhe (Ginger) Jiang, Hong-Ying Wei
Group: Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary:  This protocol is a continuation of the previous protocol (dx.doi.org/10.17504/protocols.io.m4yc8xw) for viral metagenomic researches with the tissues of Molluscs, such as abalone, oyster, clam,  scallop and so on. This maybe also applicable for other animals, such as shrimp and fish, based on the principle of the protocol. But we havn't verified this idea yet.

Proper citation: Jing-Zhe (Ginger) Jiang, Hong-Ying Wei 2018. Nucleic Acid Extraction, Amplification and Library Construction for Viral Metagenomic Sequencing.. protocols.io https://dx.doi.org/10.17504/protocols.io.m5vc866 Copy   

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Authors: Elizabeth Fozo

Proper citation: Elizabeth Fozo 2021. Cytochrome C Assay_small_volume. protocols.io https:// Copy   

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Authors: Angelica S. Gouveia, Lucas L. Lima, Flaviane S. Coutinho, Juliano M. Rodrigues, Valquiria J. M. Pinheiro, Maria Eduarda S. Ramos, Camilo E. Vital, Claudia S.L. Pontes, Daniella P. Pinheiro, Pedro M. Vidigal, Edvaldo Barros, Humberto J O Ramos
Group: Metabolomics Protocols & Workflows
Summary: Plants produce a variety of compounds with diverse structure and abundance that play an important role in the development, growth and response to the environment. Metabolomics involves studies of a great number of metabolites, which are small molecules present in biological systems.Metabolite profiles can be obtained from several samples by LC/MS (liquid chromatography coupled to mass spectrometry). However, the large number of ions detected for each run from different treatments and replicates require automatic processing for both spectra (metabolite identification and quantification) and global analysis of the metabolome. Thus, this protocol describes in detail, step-by-step, the comparison of the metabolite profiles from LC/MS data for determination of dysregulated pathways, as well as for Exploratory Statistical and Functional Enrichment Analyzes. . .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Angelica S. Gouveia, Lucas L. Lima, Flaviane S. Coutinho, Juliano M. Rodrigues, Valquiria J. M. Pinheiro, Maria Eduarda S. Ramos, Camilo E. Vital, Claudia S.L. Pontes, Daniella P. Pinheiro, Pedro M. Vidigal, Edvaldo Barros, Humberto J O Ramos 2019. METABOLIC PATHWAY ANALYSIS BY LIQUID CHROMATOGRAPHY (UHPLC) COUPLED TO HIGH RESOLUTION MASS SPECTROMETRY (LC/MS). protocols.io https://dx.doi.org/10.17504/protocols.io.8vjhw4n Copy   

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Authors: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: Rapid advancements in long-read sequencing technologies have transformed sequencing read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles, insects and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale at low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with eucalypts, acacias, rice, themeda, wheat, wheat rusts, various other fungi, geckos, skinks, ticks, ladybird beetles, caterpillars and E. coli.

Proper citation: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger 2021. High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bss7nehn Copy   

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Authors: 宏亮 董
Group: 2019 iGEM NEFU_China
Summary: The concentration of the agarose gel is 1%.

Proper citation: 宏亮 董 2019. Agarose Gel Electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.7h3hj8n Copy   

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Authors: New England Biolabs
Group: Grupo de Estudos de Leveduras Cervejeiras

Proper citation: New England Biolabs 2015. SOB Media. protocols.io https://dx.doi.org/10.17504/protocols.io.crrv55 Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community
Summary: This is a collection of protocols for: "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. Background and Rationale (Part 1 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj5tkq6n Copy   

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Authors: Luciano Martelotto
Summary: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.

Proper citation: Luciano Martelotto 2021. S.O.F protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® discontinuous gradient . protocols.io https://dx.doi.org/10.17504/protocols.io.brdcm22w Copy   

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Authors: Jorge Carlos Ruiz Ruiz
Summary: Flavonoid content determination using the aluminum chloride method.

Proper citation: Jorge Carlos Ruiz Ruiz 2018. Determination of flavonoid content. protocols.io https://dx.doi.org/10.17504/protocols.io.sfnebme Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for human intestine cell dissociation. 

Proper citation: Andrew Potter 2018. Intestine cell dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nzjdf4n Copy   

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Authors: Jacob Beal, Cheryl Telmer, Richard Tennant, Paul Rutten
Group: iGEM Measurement
Summary: This protocol can be applied to any strain of cell that can be safely run through a flow cytometer. For clarity, we have written it assuming E. coli DH5-alpha; to apply the protocol to another cell type, substitute the other cell type for any place where the protocol says [E. coli DH5-alpha].This protocol has been written for measurement of GFP, YFP, or other yellow/green fluorescent proteins into MEFL units. To apply it to fluorescent proteins of other colors:Replace BBa_J364001 with a construct for strong expression of the other protein. For blue proteins (e.g., mTagBFP), measure with 405nm excitation and 450nm/50nm emission filter. Units will be MEC30.For red/orange proteins (e.g., mCherry), measure with 561nm excitation and 610nm/20nm or 620nm/15nm emission filter.  Units will be MEPTR.For far-red / near-infrared proteins (e.g., IRFP), measure with 635nm excitation and 780nm/60nm or 750nm long-pass (LP) emission filter. Units will be MEAPCY7.For blue proteins (e.g., mTagBFP), measure with 405nm excitation and 450nm/50nm emission filter. Units will be MEC30.For red/orange proteins (e.g., mCherry), measure with 561nm excitation and 610nm/20nm or 620nm/15nm emission filter.  Units will be MEPTR.For far-red / near-infrared proteins (e.g., IRFP), measure with 635nm excitation and 780nm/60nm or 750nm long-pass (LP) emission filter. Units will be MEAPCY7.For red/orange proteins (e.g., mCherry), measure with 561nm excitation and 610nm/20nm or 620nm/15nm emission filter.  Units will be MEPTR.For far-red / near-infrared proteins (e.g., IRFP), measure with 635nm excitation and 780nm/60nm or 750nm long-pass (LP) emission filter. Units will be MEAPCY7.To apply the protocol to multiple colors, add a positive process control for each color and use one of the tools on the iGEM Measurement Resources page to compensate measurements for spectral overlap.This protocol can be combined with bead-based cell size calibration.

Proper citation: Jacob Beal, Cheryl Telmer, Richard Tennant, Paul Rutten 2019. iGEM Calibration Protocol - Flow Cytometry Fluorescence. protocols.io https://dx.doi.org/10.17504/protocols.io.2pcgdiw Copy   

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Authors: Sarah Reiling, Shu-Huang Chen, Anne-Marie Roy, Josh Quick, Ioannis Ragoussis
Group: Coronavirus Method Development Community, McGill Genome Centre
Summary: SARS-CoV-2 McGill Nanopore sequencing protocol SuperScript IV_42C_ArticV3

Proper citation: Sarah Reiling, Shu-Huang Chen, Anne-Marie Roy, Josh Quick, Ioannis Ragoussis 2020. SARS-CoV-2 McGill Nanopore sequencing protocol SuperScript IV_42C_ArticV3. protocols.io https://dx.doi.org/10.17504/protocols.io.bjajkicn Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol outlines the analysis used to generate input files for CoNet for the phage-bacteria network. Based on methods from the following publication:Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script R15: CoNet Analysis - Formatting Relative Abundance Files. protocols.io https://dx.doi.org/10.17504/protocols.io.ejpbcmn Copy   

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Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community

Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters. protocols.io https://dx.doi.org/10.17504/protocols.io.8g5hty6 Copy   

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Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2019. Long staining procedure of nuclei in Euplotes crassus using DAPI. protocols.io https://dx.doi.org/10.17504/protocols.io.2akgacw Copy   

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Authors: James Thornton Jr
Group: Metafunc Course 2017
Summary: This protocol details the steps to annotate Anvi'o gene calls for function using Uproc. 

Proper citation: James Thornton Jr 2017. Annotate gene function with Uproc. protocols.io https://dx.doi.org/10.17504/protocols.io.kt8cwrw Copy   

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