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Authors: Promega, Trevor Wagner
Group: Promega
Summary: Protocol for PCR Cloning with Blue/White Selection and Easy Insert Excision using pGEM®-T Easy Vector Systems.(For use with A3600, A3610, A1360, or A1380.)  

Proper citation: Promega, Trevor Wagner 2018. PCR Cloning with Blue/White Selection and Easy Insert Excision. protocols.io https://dx.doi.org/10.17504/protocols.io.k5kcy4w Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process for diaminobenzidine (DAB) detection of biocytin filled cells. This protocol is optimized for use with brain slices cut at 350 µm thick, in which cells are first filled with biocytin (i.e., post-electrophysiological recording), fixed in 4% PFA/2.5% glutaraldehyde, and transferred to PBS until ready to stain.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. DAB Detection of Biocytin Labeled Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bep5jdq6 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.bgnbjvan Copy   

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Authors: Philipp Engel
Summary: This protocol is used to prepare stock solutions for a M9-based ‘minimal’ medium containing trace metals, vitamins, and casamino acids. Pollen extract or other carbon sources can be added to promote growth of bee gut bacteria (proteobacteria).

Proper citation: Philipp Engel 2017. M9 base medium for bee gut bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.kdqcs5w Copy   

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Authors: Edgar Andrade-Lotero, Robert Goldstone
Group: Universidad del Rosario
Summary: The task is a two-player game in which players interact with 64 tiles arranged in an 8x8 grid. The grid can either hide a unicorn beneath one of the tiles or else it can be absent from the grid. Either event can occur with equal probability. At the beginning of each round, the computer chooses whether or not there is a unicorn, and if there is one, it randomly chooses a tile---each one having an equal probability of being chosen---and places the unicorn beneath it. Then, players have to guess whether there is a unicorn or if it is absent from the grid by uncovering tiles one at a time, with both players uncovering tiles simultaneously, in order to see what lies beneath them. What tiles have been uncovered and whether there is or not a unicorn is only known to the player that uncovers these tiles. However, in the event that both players uncover the same tile, it changes its color and both players can immediately see this. At any time during the round, each player can make a guess as to whether the unicorn is present or absent from the grid. The other player will know this information and they can use it to inform their own guess. The round ends when both players announce that their guess is a final decision, and then they are shown their individual scores from the round. The score depends on whether the player's guess is correct (32 points) or incorrect (-64 points), subtracting the number of tiles that were uncovered by both players.

Proper citation: Edgar Andrade-Lotero, Robert Goldstone 2021. Seeking the unicorn. protocols.io https://dx.doi.org/10.17504/protocols.io.bts9nnh6 Copy   

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Authors: Lauren Krausfeldt
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: RNA extraction from water collected on Sterivex. The DNeasy PowerWater Sterivex kit is used and modified specifically for RNA. Also includes modifications for samples preserved in RNAlater.

Proper citation: Lauren Krausfeldt 2017. RNA Extraction from Sterivex filters . protocols.io https://dx.doi.org/10.17504/protocols.io.gmkbu4w Copy   

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Authors: Honghao Zhao, Jasmine Chong, Rong Tang, Li Li, Jianguo Xia, Dapeng Li
Group: Metabolomics Protocols & Workflows
Summary: The ultra-high density intensive farming model of grass carp (Ctenopharyngodon idellus) may elicit growth inhibition, decline flesh quality and increase disease susceptibility of fish. The quality degradation and excessive fat accumulation in cultured C. idellus have long been attributed to possible alterations in the lipid metabolism of fish muscle tissues as a result of over-nutrition from artificial diets. To investigate the effects of different diets on fish muscle quality, a large-scale metabolomics study was performed on 250 tails of C. idellus. The experimental fish were divided into four groups based on sex and diets - female artificial feed (FAF), female grass feed (FGF), male artificial feed (MAF) and male grass feed (MGF). After 113 days rearing period, the AF group showed significantly higher total mass of muscle fat (P Our results suggested that grass is a better source of diet fatty acid and protein when compared to artificial feed. Grass feeding could effectively lower triglycerides in serum, reduce fat accumulation and alter lipid compositions in fish muscle by increasing the concentrations of n-3 UFAs, leading to better nutrition and health.

Proper citation: Honghao Zhao, Jasmine Chong, Rong Tang, Li Li, Jianguo Xia, Dapeng Li 2018. Protocols supporting "Metabolomics Investigation of Dietary Effects on Flesh Quality in Grass Carp (Ctenopharyngodon idellus)". protocols.io https://dx.doi.org/10.17504/protocols.io.smhec36 Copy   

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Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: This SOP describes sample extraction and sample preparation for primary metabolism profiling by gas chromatography / time of flight mass spectrometry (GCTOF)References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ

Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Sample preparation for GCTOF analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ytjfwkn Copy   

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Authors: Lisa Mesrop, Sarah Hu
Group: Caron Lab - Protistan Ecology
Summary: Protocol for PCR amplification of extracted cDNA or DNA and downstream library preparation for MiSeq sequencing.See other related protocols for extraction of RNA and/or DNA from environmental samplesThis protocol is specific to 18S rRNA gene tag sequencing targeting to V4 hypervariable region. We use V4 primers with Illumina adapters that then anneal to the P5 & P7 Illumina-specific indices. This barcoding step allows us to multiplex many samples for MiSeq sequencing. The V4 region is ~400bp, so we typically do MiSeq 250x250bp PE sequencing. 

Proper citation: Lisa Mesrop, Sarah Hu 2017. 18S V4 tag sequencing PCR amplification and library prep (Illumina). protocols.io https://dx.doi.org/10.17504/protocols.io.hdmb246 Copy   

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Authors: dkt
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: dkt 2020. u-Smell-it olfactory test  . protocols.io https://dx.doi.org/10.17504/protocols.io.bkxkkxkw Copy   

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Authors: Rafal Donczew, Amélia Lalou, Steven Hahn
Summary: ChEC-seq and other nuclease-based methods such as Cut&Run map protein locations on DNA by targeting nuclease activity to specific transcription factors and mapping the resulting DNA cleavages (Schmid et al. 2004; Skene and Henikoff 2017; Zentner et al. 2015). For ChEC-seq, yeast cells expressing a protein-micrococcal nuclease (MNase) fusion are permeabilized, MNase is activated by the addition of calcium, and the resulting DNA fragments are mapped. Potential advantages of this approach include avoiding non-specific protein-DNA crosslinking in highly transcribed regions, efficient mapping of factors that do not directly bind DNA and, more sensitive detection of protein-DNA interactions. We optimized the original ChEC-seq protocol to minimize non-specific DNA cleavage, avoid over digestion at authentic binding sites, and efficiently assay large numbers of factors. We also created a robust data analysis pipeline that incorporates peak calling to map binding sites and quantitative analysis, based on utilization of spike-in DNA, to compare factor-DNA binding under different conditions. We used this modified approach to map genome-wide distributions of the transcription coactivators TFIID and SAGA (Donczew et al. 2020) as well as transcription factors Abf1 and Rap1 (Donczew et al, submitted to Mol Cell).ReferencesDonczew R, Warfield L, Pacheco D, Erijman A, Hahn S. 2020. Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.eLife 9: e50109.Schmid M, Durussel T, Laemmli UK. 2004. ChIC and ChEC; genomic mapping of chromatin proteins.Mol Cell 16: 147–157.Skene PJ, Henikoff S. 2017. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.eLife 6: e21856.Warfield L, Ramachandran S, Baptista T, Devys D, Tora L, Hahn S. 2017. Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID.Mol Cell. 68:118-129Zentner GE, Kasinathan S, Xin B, Rohs R, Henikoff S. 2015. ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.Nat Commun 6: 8733.

Proper citation: Rafal Donczew, Amélia Lalou, Steven Hahn 2020. An improved ChEC-seq method for mapping the genome-wide binding of S. cerevisiae transcription factors. protocols.io https://dx.doi.org/10.17504/protocols.io.bizgkf3w Copy   

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Authors: Daisy Perez-Brito
Summary: The importance of fungal species differentiation is critical for plant pathology research and disease control purposes. Traditionally, fungal species have been identified by a range of cultural and morphological characteristics, such as conidial morphology, presence or absence of setae, fungicide sensitivity, and colony color and growth rate. Although valuable, these criteria alone are not always adequate as morphological characteristics may vary under different environmental conditions. Among other techniques, sequence analyses of the internal transcribed spacer (ITS) regions, including the 5.8S rDNA, has been used extensively to identify fungal species. This region is the most widely sequenced DNA region of fungi and has been employed as the universal fungal barcode sequence. It is very useful for molecular systematics at species to genus level and within species.This protocol is useful for a primary molecular and accurate identification of any fungal species using the sequence analyses of the ITS-5.8S rDNA region

Proper citation: Daisy Perez-Brito 2018. MOLECULAR IDENTIFICATION OF PHYTOPATHOGENIC FUNGI. protocols.io https://dx.doi.org/10.17504/protocols.io.shteb6n Copy   

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Authors: Don Cooper
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: Don Cooper 2020. COVIDscanDX LAMP. protocols.io https://dx.doi.org/10.17504/protocols.io.bk4dkys6 Copy   

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Authors: DNA Pipelines R&D, Benjamin Farr, Diana Rajan, Emma Betteridge, Lesley Shirley, Michael Quail, Naomi Park, Nicholas Redshaw, Iraad Bronner, Louise Aigrain, Scott Goodwin, Scott Thurston, Stefanie Lensing, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K. Jackson, Alex Alderton, Ian Johnston
Group: Coronavirus Method Development Community, COG-UK
Summary: This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing 400nt amplicons tiling the viral genome using V3 nCov-2019 primers (ARTIC). This is followed by library construction, equivolume pooling of samples and quantitation, prior to sequencing on the Illumina NovaSeq. It is adapted from the nCov-2019 sequencing protocol which can be found here: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w.

Proper citation: DNA Pipelines R&D, Benjamin Farr, Diana Rajan, Emma Betteridge, Lesley Shirley, Michael Quail, Naomi Park, Nicholas Redshaw, Iraad Bronner, Louise Aigrain, Scott Goodwin, Scott Thurston, Stefanie Lensing, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K. Jackson, Alex Alderton, Ian Johnston 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bgxjjxkn Copy   

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Authors: Mikhail Murzin

Proper citation: Mikhail Murzin 2019. asdsda. protocols.io https://dx.doi.org/10.17504/protocols.io.747hqzn Copy   

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Authors: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones
Group: RENE FLORES

Proper citation: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones 2021. EXTRACTS PRODUCTION AND FRACTIONATION. protocols.io https://dx.doi.org/10.17504/protocols.io.br2cm8aw Copy   

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Authors: Liz O'Brien, Connor Tsuchida
Group: The Center for Genome Editing and Recording

Proper citation: Liz O'Brien, Connor Tsuchida 2019. DNA/RNA Radiolabeling Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8dshs6e Copy   

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Authors: Ruth Timme, Emma Griffiths, Heather Blankenship, Erin Young, Duncan MacCannell, Stacia Wyman
Group: GenomeTrakr, Coronavirus Method Development Community, PHA4GE
Summary: PURPOSE: This is a SARS-CoV-2 specific protocol that covers the steps needed to establish a new NCBI submission environment for your laboratory, including the creation of new BioProject(s) and submission groups. Once these are step up, the protocol then walks through the process for submitting raw reads to SRA and sample metadata to BioSample through the Submission portal. For new submitters, there's quite a bit of groundwork that needs to be established before a laboratory can start its first data submission. We recommend that one person in the laboratory take a few days to get everything set up in advance of when you expect to do your first data submission. If you need a pipeline for frequent or large volume submissions, follow Step 1 in the SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject to get your NCBI submission environment established, then contact [email protected] to set up an account for submitting through the API. These protocols cover submission using NCBI's Submission Portal web-interface.Complete in order (1 then 2):1. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject (included protocol)Step-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort.SARS-CoV-2 raw data submission to SRA (Sequence Read Archive) and metadata to BioSample.2. SARS-CoV-2 NCBI assembly submission protocol: GenBank Required: established BioProject and BioSamplesSubmit SARS-CoV-2 assemblies to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data.2. SARS-CoV-2 NCBI assembly submission protocol: GenBank Required: established BioProject and BioSamples

Proper citation: Ruth Timme, Emma Griffiths, Heather Blankenship, Erin Young, Duncan MacCannell, Stacia Wyman 2020. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject. protocols.io https://dx.doi.org/10.17504/protocols.io.bf7bjrin Copy   

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Authors: Yashodara Abeykoon, Adrienne Cho, Celina Juliano

Proper citation: Yashodara Abeykoon, Adrienne Cho, Celina Juliano 2019. Hydra Dissociation/Reaggregation . protocols.io https://dx.doi.org/10.17504/protocols.io.6tyhepw Copy   

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Authors: Hossein Tarrahimofrad, Amir Meimandipour, Sareh Arjmand, Mohammadtaghi Beigi Nassiri, Ehsan Jahangirian, Hossein Tavana, Javad Zamani, Somayyeh Rahimnahal, Saeed Aminzadeh
Summary: The prepared protein and protein-ligand complexes (protease-SDS and protease-tween complexes) were used in MD simulation, performed using GROMACS v 4.6.5 with CHARMM36 all-atom force field. The CGenFF server (https://cgenff.umaryland.edu/) provides topologies and parameters of ligands compatible with the CHARMM36 all-atoms force field. Protein and protein-ligand complexes were soaked in a cubic box of water molecules, and the charges on the protein were neutralized by the addition of Na+ and Cl- ions. The energy of the system was minimised using the steepest descent algorithm to eliminate bad contact and clashes. The NVT and NPT ensembles were used during the equilibration to achieve the desired temperature (373.15 K for protease and 300 K for protease-ligand complex) and pressure (1 bar) for 100 ps and restraint forces of 1000 kJ/mol. Finally, 40 ns MD run were performed in triplicate after releasing all restraints. All bonds were constrained by the LINCS algorithm.

Proper citation: Hossein Tarrahimofrad, Amir Meimandipour, Sareh Arjmand, Mohammadtaghi Beigi Nassiri, Ehsan Jahangirian, Hossein Tavana, Javad Zamani, Somayyeh Rahimnahal, Saeed Aminzadeh 2020. Molecular dynamics simulation (Protein-Ligand). protocols.io https://dx.doi.org/10.17504/protocols.io.bgcajsse Copy   

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