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Authors: Daniel Vaulot
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Roscoff Culture Collection
Summary: Protocol to record videos on SBR Microscope

Proper citation: Daniel Vaulot 2019. Microscope video recording. protocols.io https://dx.doi.org/10.17504/protocols.io.k24cygw Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. Diabetic Complication:

Proper citation: Eva Feldman 2019. Biochemical Measures of Neuropathy -- Glutathione S-Transferase. protocols.io https://dx.doi.org/10.17504/protocols.io.3sbgnan Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the synthesis of first-strand cDNA from total RNA using the Invitrogen SuperScript III Reverse Transcriptase system.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. cDNA Synthesis. protocols.io https://dx.doi.org/10.17504/protocols.io.besejebe Copy   

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Authors: Umed Boltaev, Yves Meyer
Summary: KIRA-ELISA is used for quantitave detection of phosphorylation level of tyrosine kinase receptors. The protocol could be used for many different receptors. In our work, this protocol has been successful for TrkB and FGFR1 receptors. The underlying principle of the assay is capturing the receptor and detecting phosphorylated tyrosine on the intracellular domain of the receptor using pan phospho-tyrosine antibody. Since tyrosine kinase receptor are phosphorylated at multiple tyrosine residues, pan phospho-tyrosine antibody detects any possible antigen on the receptor, resulting in more sensitive assay. Our assay is based on previously published bioassay by M. Sadick et. al. and M. Cazorla et. al.

Proper citation: Umed Boltaev, Yves Meyer 2018. Sandwich KIRA-ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rprd5m6 Copy   

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Authors: Emma Ganley, Anita Broellochs, Lenny Teytelman
Summary: A list to advise on the different elements that are possible to include as part of your protocol in protocols.io. If possible, please include these in the PDF of the protocol that you submit for us to import. Our editors will then be able to include those as part of your protocol.Ideally your protocol PDF will show the protocol as a step-by-step 'recipe' style protocol. Please try to lay it out this way rather than sending us the materials and methods text from a manuscript as that will be hard to interpret as a step-by-step protocol, and crucially, it will not include a lot of the important details.Please note that if you have a protocol PDF ready to submit, but do not yet have these additional elements ready, you can still go ahead and submit the PDF and there will be a chance to provide the rest to us later, or add them yourself. Once we transfer ownership of the protocol back to you, you'll be able to further edit the protocol prior to sharing or publishing (you can add thumbnail image, photos or videos etc.)

Proper citation: Emma Ganley, Anita Broellochs, Lenny Teytelman 2021. What to include in the PDF of your protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.br39m8r6 Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Checking DNA Concentration with Agarose Gel. protocols.io https://dx.doi.org/10.17504/protocols.io.c35yq5 Copy   

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Authors: John Tyson
Summary: Size Selective Precipitation of DNA using PEG & SaltWe and others have been thinking about the possibility of providing DNA size selection / clean up / and removal of expensive reagents in the library preparation process for a little while now. There are a number of possible routes but we have focused more recently on Polyethylene Glycol (PEG) and salt precipitation of DNA as it is cheap, non-toxic and known to be compatible with the existing library preparation process. One of the early demonstrations of combining PEG and salt to selectively size fractionate DNA by selective precipitation was by Lis & Schleif in 1975 ( Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. NAR 2:(3) p383). We have been screening different sizes and concentrations of PEG combined with different salt amounts to find suitable combinations for removal of short “contaminating” DNA fragments. This is useful for situations where you are not starting with a DNA sample of HMW and want to target the longest DNA strands you have or want to try and remove shorter fragments produced during a preparation.Shown below are some of our current best combinations but the hunt goes on, with changes to type and concentration of PEG, and different cations with different charge densities. The commercially available Circulomics buffers perform a similar task, and are performing a little better at this stage. We do not know what exactly has been used in their solutions.One advantage of knowing that PEG/NaCl can be used in this fashion came with the realisation that given ligation buffers often use high PEG concentrations to crowd the DNA fragments we could probably use straight NaCl addition into the ligation reaction once complete to precipitate our adapted DNA. This is shown below with testing on a Lambda+DNA ladder sample and the ONT LNB buffer +/- 600 mM NaCl (There is also the hint there that some level of HMW DNA is even coming out of solution without the salt addition…….). This idea together with PEG/NaCl precipitation when coming out of the LSK109 library End-Prep reaction was taken and used to develop the Bead-free LSK109 ligation prep for ultra-long DNA detailed below in the next section.If people feel so inclined I can provide further details on the current matrix of PEG size / concentrations and salt type / concentrations we have looked at so far and then we can perhaps speed up the search. There are obviously other possibilities that can be explored around size selection using charged polymers and higher charge density ion induced precipitation. Only so many hours in the day :o)).

Proper citation: John Tyson 2020. Size Selective Precipitation of DNA using PEG & Salt. protocols.io https://dx.doi.org/10.17504/protocols.io.7erhjd6 Copy   

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Authors: Monika Ruwaimana, Farid Dahdouh-Guebas, Behara Satyanarayana1

Proper citation: Monika Ruwaimana, Farid Dahdouh-Guebas, Behara Satyanarayana1 2018. The advantages of using drones over space-borne imagery in the mapping of mangrove forests: Data Analysis Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.qh7dt9n Copy   

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Authors: Sarah Hessen-Schmidt

Proper citation: Sarah Hessen-Schmidt 2016. Wash Buffer (10mM Tris-HCl pH 7.5, 80% EtOH). protocols.io https://dx.doi.org/10.17504/protocols.io.fjmbkk6 Copy   

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Authors: Integrated Islet Distribution Program
Group: Integrated Islet Distribution Program, Integrated Islet Distribution Program and Human Islet Phenotyping Program
Summary: This Standard Operating Procedure is adapted from the work of the 'National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities'' following the SOP cited in the document 'Purified Human Pancreatic Islet: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ) – Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'This SOP defines the assay method for quantitative and qualitative determination in the identification of human isolated islet preparations, which include endocrine and exocrine tissue, for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP). This protocol is written to assist the participating islet isolation centers and investigators who are part of this program.Dithizone (diphenyl thiocarbazone; DTZ) is an organic chemical that chelates the zinc in the insulin granules present in the beta cells of the pancreatic islets. The islet cells are stained red while the acinar cells remain unstained.DTZ staining is used as a lot release and as an in-process assay: (i)Lot release testing: DTZ staining is used to identify islets and to determine the quantity and quality of the final islet product. Islet quantity is expressed as the number of islet equivalents (IEQ), which is calculated based on the number and diameter of the islets present in the preparation, mathematically corrected for islet volume. (ii)In-process testing: DTZ staining is used to identify islets and to assess the effectiveness of the digestion, isolation and purification processes. The quality of the preparations is expressed as percent islet purity, and percent trapped islets. Islet quantity (IEQ) is also assessed.

Proper citation: Integrated Islet Distribution Program 2020. Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ). protocols.io https://dx.doi.org/10.17504/protocols.io.bk5vky66 Copy   

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Authors: Rosa I Ramos-Arredondo, Blanca E, Diane Gendron, J. Francisco Gallegos-Funes, Dante Mújica-Vargas, J.B. Rosas-Fernández
Summary: Photoidentification is a method used by biologists to classify animals primarily to track endangered species. Different methods of identification, either manual or computational, have been developed since the 1990s, which until now have remained in use. However, these methods can introduce a factor of subjectivity when classifying an individual animal, creating uncertainty or inaccuracy in the results obtained as a result of the human criteria involved. For this reason, one of the main objectives in photoidentification is to implement an automated mechanism that is free of biases, is portable, and is easily used. The main aim of this work is to develop an autonomous and portable photoidentification system through the optimization of image classification algorithms that have high statistical dependence, with the goal of classifying images of the dorsal fin of the blue whale through offline information processing on a mobile device. The new proposed methodology is based on the Scale Invariant Feature Transform (SIFT) that, in conjunction with statistical discriminators such as the variance and the standard deviation, fits the extracted data and selects the closest pixels that make up the edges of the dorsal fin of the blue whale. In this way, we ensure the elimination of the most common external factors that could affect the quality of the image, thus avoiding the elimination of relevant sections of the dorsal fin. The blue whale photoidentification method presented in this work has been developed at the Instituto Politécnico Nacional on the coast of Baja California Sur. The results shown have been validated qualitatively and quantitatively in terms of sensitivity, specificity and accuracy on the Jetson Tegra TK1 mobile platform. The presented solution balances the results obtained with the computational cost of the execution of the algorithm, and yields a portable system that could be beneficial for field studies.

Proper citation: Rosa I Ramos-Arredondo, Blanca E, Diane Gendron, J. Francisco Gallegos-Funes, Dante Mújica-Vargas, J.B. Rosas-Fernández 2020. PhotoId-Whale: blue whale dorsal fin classification for mobile devices. protocols.io https://dx.doi.org/10.17504/protocols.io.be2rjgd6 Copy   

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Authors: Curtis Huttenhower
Group: VERVE Net, Huttenhower Lab
Summary: In this protocol we will show how to taxonomically profile 20 metagenomic shotgun samples Human Microbiome Project (HMP). Specifically, we will look at 10 samples from the buccal mucosa and 10 from the tongue dorsum. Please note these 20 samples are not necessarily an unbiased selection of microbiomes from the over 200 buccal mucosa and tongue dorsum samples generated by the HMP.REQUIREMENTS: BowTie2 installed and in the system path, Mercurial, basic unix commands (wget, tar).The size of the 20 samples (60GBs) can make their download quite slow. For this reason we are also providing the overall output of this protocol (few KBs) available for download so that one can start the tutorial from MetaPhlAn output merge and visualization if needed.

Proper citation: Curtis Huttenhower 2016. MetaPhlAn profiling of 20 HMP samples. protocols.io https://dx.doi.org/10.17504/protocols.io.d9e93d Copy   

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Authors: Mustafa Munawar

Proper citation: Mustafa Munawar 2020. Protocol for RPA-PCR Couple. protocols.io https://dx.doi.org/10.17504/protocols.io.bifrkbm6 Copy   

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Authors: James Lyon, Patrick Macdonald, Jocelyn Manning Fox
Group: Human Cell Atlas Method Development Community

Proper citation: James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Human Islet Quantification and Purity Assessment. protocols.io https://dx.doi.org/10.17504/protocols.io.si3ecgn Copy   

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Authors: Franziska Aron, Gunnar Neumann, Christina Warinner, Guido Brandt
Group: WarinnerGroup
Summary: Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols.This protocol does not include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).

Proper citation: Franziska Aron, Gunnar Neumann, Christina Warinner, Guido Brandt 2020. Non-UDG treated double-stranded DNA library preparation for Illumina sequencing of ancient dental calculus . protocols.io https://dx.doi.org/10.17504/protocols.io.bqcsmswe Copy   

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Authors: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: We aim to develop high resolution, chemically informative imaginig methodologies for building an atlas of human organs, such as the kidney.Scope:Provide an overview of the methods used by the Vanderbilt Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.

Proper citation: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2019. VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN). protocols.io https://dx.doi.org/10.17504/protocols.io.7hhhj36 Copy   

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Authors: Tingting Xiang, Arthur Grossman
Group: Aiptasia-Symbiodiniaceae Model System
Summary: Dinoflagellates are unicellular algae that can have photosynthetic or nonphotosynthetic lifestyles. Dinoflagellates in the genus Symbiodinium can enter endosymbiotic associations with corals, providing the metabolic basis for the highly productive and biologically diverse coral-reef ecosystems (Hoegh-Guldberg, 1999), as well as with other cnidarians, including sea anemones and jellyfish, and noncnidarian hosts (Trench, 1993; Lobban et al., 2002; Mordret et al., 2016).Here I describe a protocol for isolating total DNA from Symbiodinium cells.

Proper citation: Tingting Xiang, Arthur Grossman 2018. DNA Extraction from Symbiodinium Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.qhbdt2n Copy   

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Authors: John Tyson
Summary: Modified LSK109 ligation prep using needle shear and bead purificationUsing HMW DNA purified as above we have been performing 29G and 26G needle shears to produce fragmentation to around 18Kb and 34kb respectively (reported read N50’s after basecalling). Also by going into the library preparations with 5-10ug of DNA have been able to produce enough material to provide up to 10 library loads from a single prep. We have not been performing the FFPE repair during the end-prep step, and have been diluting the sample at the cleanup points in the protocol with EB to prevent bead clumping during 0.4-0.5x AMPureXP purifications when DNA fragments are large and at high concentrations. Shown below are the results from a batch of 12 runs we performed using this approach.

Proper citation: John Tyson 2020. Modified LSK109 ligation prep with needle shear and bead clean up. protocols.io https://dx.doi.org/10.17504/protocols.io.7emhjc6 Copy   

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Authors: Vincent Bielinski

Proper citation: Vincent Bielinski 2020. Small-scale Expression and Solubility Testing of Proteins in BL21 E. coli . protocols.io https:// Copy   

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Authors: Ken Youens-Clark
Group: Hurwitz Lab
Summary: RefSeqMash uses the MinHash (http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0997-x) algorithm to characterize the genera/species/strains present in metagenomic samples by comparing the reads to ~11K known RefSeq genomes.

Proper citation: Ken Youens-Clark 2016. RefSeqMash 0.0.2. protocols.io https://dx.doi.org/10.17504/protocols.io.fy7bpzn Copy   

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