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Authors: Noreen Wauford

Proper citation: Noreen Wauford 2016. Hemolysis Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.fxkbpkw Copy   

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Authors: Peter Vangheluwe, Shaun Martin, Mujahid Azfar
Summary: The 4-methylumbelliferyl heptanoate (MUH) assay is a A fluorimetric assay used to study cell-mediated cytotoxicity. The principle behind this assay employs the activity of esterases found in viable cells. These esterases then hydolyse the fluorochrome in the MUH reagent which can then be read using a plate reader.

Proper citation: Peter Vangheluwe, Shaun Martin, Mujahid Azfar 2020. 4-methylumbelliferyl heptanoate (MUH) - Cell Viability Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bazjif4n Copy   

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Authors: Snehadri Sinha
Group: Aalto-Helsinki 2016
Summary: PCR using Phusion HotStart II Hifi Mastermix (based on ThermoFisher Scientific manufacturer protocol).

Proper citation: Snehadri Sinha 2017. Phusion HotStart II Hifi PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.gzjbx4n Copy   

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Authors: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro
Group: Ribeiro Lab
Summary: This is a collection of methods and protocols from the manuscript: Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.

Proper citation: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro 2017. Methods and protocols from Goncalves et al. (2017) for manipulating the diet and the microbiome of Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.hdtb26n Copy   

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Authors: Bei Wang, Daniel Asarnow, Wen-Hsin Lee, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Hwee Ching Tan, Chia-Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Yuanyu Hu, Shirley Gek Kheng Seah, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa-Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang
Group: Coronavirus Method Development Community
Summary: In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Proper citation: Bei Wang, Daniel Asarnow, Wen-Hsin Lee, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Hwee Ching Tan, Chia-Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Yuanyu Hu, Shirley Gek Kheng Seah, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa-Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang 2020. Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization. protocols.io https://dx.doi.org/10.17504/protocols.io.bi35kgq6 Copy   

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Authors: John Rutledge
Group: Mouse Metabolic Phenotyping Centers
Summary: One of the three indices of arterial function that are compromised to a varying degree in individuals with cardiovascular disease is vascular permeability. This assay measures vascular permeability (as flux of labeled large molecular weight molecules: i.e. albumin or dextran) and lipid permeability (as flux of labeled lipid) in coronary or carotid arteries.

Proper citation: John Rutledge 2019. UC Davis - Microvascular Permeability and Lipoprotien Flux. protocols.io https://dx.doi.org/10.17504/protocols.io.56vg9e6 Copy   

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Authors: Etienne Laliberté
Group: Plant Functional Ecology Lab, Canadian Airborne Biodiversity Observatory
Summary: Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure leaf water content and specific leaf area, using the WinFOLIA™ software (Régent Instruments). These leaf area and water measurements are done on a subset of leaves from the same bulk leaf sample used to measure leaf spectral reflectance and transmittance. Briefly, after removing their petioles, fresh leaves are weighed, rehydrated for 6 h, scanned for total leaf area and weighed again; they are then oven-dried at 65 ºC for 72 h, and weighed one last time. This allows us to measure leaf dry matter content and its complement, leaf water content, as well as leaf relative water content. Leaf area measurements are used to estimate specific leaf area, a key functional trait central to the leaf economics spectrum. Specific leaf area allows us to estimate equivalent water thickness and to convert concentrations of foliar biochemical constitutents from a leaf mass to a leaf area basis.

Proper citation: Etienne Laliberté 2018. Measuring specific leaf area and water content. protocols.io https://dx.doi.org/10.17504/protocols.io.p3tdqnn Copy   

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Authors: Peter J. Skene, Steven Henikoff
Group: Human Cell Atlas Method Development Community
Summary: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench.In summary, CUT&RUN has several advantages over ChIP-seq: (1) The method is performed in situ in non-crosslinked cells and does not require chromatin fragmentation or solubilization; (2) The intrinsically low background allows low sequence depth and identification of low signal genomic features invisible to ChIP; (3) The simple procedure can be completed within a day and is suitable for robotic automation; (4) The method can be used with low cell numbers compared to existing methodologies; (5) A simple spike-in strategy can be used for accurate quantitation of protein-DNA interactions. As such, CUT&RUN represents an attractive replacement for ChIPseq, which is one of the most popular methods in biological research.

Proper citation: Peter J. Skene, Steven Henikoff 2018. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. protocols.io https://dx.doi.org/10.17504/protocols.io.mgjc3un Copy   

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Authors: Lukas Snoek, Tinka Beemsterboer
Group: Spinoza Centre, REC-L
Summary: This protocol lists all the steps necessary to run your MRI experiment/data acquisition safely and in a way that yields high-quality data. Moreover, if you use the centre's QC/preprocessing service, it lists the steps necessary to make sure we can convert the data into BIDS and run them through the QC/preprocessing pipelines.

Proper citation: Lukas Snoek, Tinka Beemsterboer 2019. During data acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.ypwfvpe Copy   

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Authors: Peng Wang, Hongjian Ni, Ruihe Wang

Proper citation: Peng Wang, Hongjian Ni, Ruihe Wang 2018. Fig 9. Effect of installation distance of shakers on weight on bit and accelerations of the drill-string. A. Change of weight on bit in the bottom hole over time. B. Maximum acceleration along the drill-string. C. Root mean square acceleration along the dr. protocols.io https://dx.doi.org/10.17504/protocols.io.kfactie Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR). protocols.io https:// Copy   

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Authors: Molly Brennan, Olivier George
Group: George Lab

Proper citation: Molly Brennan, Olivier George 2020. 4% Formaldehyde in .1M PB (FOR IMMUNO BRAINS) (1L). protocols.io https:// Copy   

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Authors: Mark Dewitt & Julia Wong
Group: Innovative Genomics Institute, CornLab
Summary: This protocol, based on published work, demonstrates how to delivery Cas9 RNP-based gene editing reagents to cultured mamallian cells by electroporation with a Lonza 4d Nucleofector. Consider consulting some of the following papers:1. RNP delivery paper upon which this work is based (Open Access):https://elifesciences.org/content/3/e047662. Paper by an IGI post-doc that details the rationale behind HDR donor design:https://www.ncbi.nlm.nih.gov/pubmed/26789497

Proper citation: Mark Dewitt & Julia Wong 2017. Cas9 RNP nucleofection for cell lines using Lonza 4D Nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.hd8b29w Copy   

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Authors: Sonia Hall

Proper citation: Sonia Hall 2016. Blocking Solution (Donkey). protocols.io https://dx.doi.org/10.17504/protocols.io.fevbje6 Copy   

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Authors: Daniel Richter
Summary: Phenol: chloroform extraction to clean up RNA ( e.g. to remove DNAse or DNAse Inactivation Reagent.) Based on phenol/chloroform protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5438.full), ethanol precipitation protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5440.full)

Proper citation: Daniel Richter 2019. RNA clean-up by phenol:chloroform. protocols.io https://dx.doi.org/10.17504/protocols.io.8y3hxyn Copy   

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Authors: Zhao-Ting Meng, Fan Cui, Xue-Ying Li, Dong-Xin Wang
Summary: Total knee arthroplasty (TKA) is an important therapy for patients with serious knee osteoarthritis in order to improve quality of life and relieve pain. But a large number of patients who undergo this surgery experience moderate to severe postoperative pain. Previously, the investigators used single femoral nerve blockade combined with patient-controlled intravenous analgesia for postoperative analgesia for patients after TKA. Although this method provides acceptable analgesia, the incidence of opioid-associated side effects is relatively high. Low-dose epidural morphine is commonly used in postoperative analgesia after cesarean section, and the effect of single dose morphine lasts more than 20 hours, with low incidences of itching, nausea, vomiting, and respiratory depression. The investigators hypothesize that, for patients undergoing TKA, the addition of low-dose epidural morphine to single femoral nerve block and intravenous opioids can improve the postoperative analgesia, reduce the consumption of intravenous opioids and decrease opioid-associated side effects.

Proper citation: Zhao-Ting Meng, Fan Cui, Xue-Ying Li, Dong-Xin Wang 2018. Epidural morphine combined with single-injection femoral nerve block for postoperative analgesia in patients after total knee arthroplasty: a randomized controlled trial. protocols.io https://dx.doi.org/10.17504/protocols.io.wanfade Copy   

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Authors: James Collins, James R. Collins, Krista Longnecker, Helen F. Fredricks, Benjamin A. S. Van Mooy
Group: Van Mooy Lab @ WHOI
Summary: The protocols in this collection were original created by Krista Longnecker and Jamie Collins for creating lipsomes to be used in lipid photo-oxidation experiments. The results of these experiments are detailed in:Collins, J. R, H. F. Fredricks, J. S. Bowman, C. P. Ward, C. Moreno, K. Longnecker, A. Marchetti, C. M. Hansel, H. W. Ducklow, and B. A. S. Van Mooy. 2018. The molecular products and biogeochemical significance of lipid photooxidation in West Antarctic surface waters. Geochimica et Cosmochimica Acta 232:244–264; doi:10.1016/j.gca.2018.04.030and in Chapter 4 of:Collins, J. R. 2017. The remineralization of marine organic matter by diverse biological and abiotic processes. Ph.D. thesis. Cambridge, Massachusetts: Massachusetts Institute of Technology, 300 pp; doi:10.1575/1912/8721

Proper citation: James Collins, James R. Collins, Krista Longnecker, Helen F. Fredricks, Benjamin A. S. Van Mooy 2019. Two-step protocol: Preparation and extrusion of phospholipid liposomes. protocols.io https://dx.doi.org/10.17504/protocols.io.zbef2je Copy   

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Authors: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao
Group: GigaScience Press, BGI, GIGA, GigaScience Press
Summary:  BGISEQ-500 is a new desktop sequencer developed by BGI. Using DNA nanoball and combinational probe anchor synthesis developed from Complete Genomics™ sequencing technologies, it generates short reads at a large scale. 

Proper citation: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao 2018. BGISEQ-500 Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.pq7dmzn Copy   

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Authors: Nikki Freed, Olin Silander
Group: Coronavirus Method Development Community
Summary: To enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than current methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit (RBK004). This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick, which produces 400 base pair amplicons and uses the Oxford Nanopore Ligation barcoding kit (LSK-109). We have increased the size of the amplicons to 1200bp and use the RAPID barcode kit (RBK004), which enables requires less time and fewer reagents than the LSK-109 protocol. The amplicons produced in this protocol could also be used for Illumina sequencing. Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.Primer sequences are here: https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharingWe can ship a small amount of pooled primers to interested labs for further testing, email [email protected] or [email protected]

Proper citation: Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon). protocols.io https://dx.doi.org/10.17504/protocols.io.bfwnjpde Copy   

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Authors: Kathy Lam

Proper citation: Kathy Lam 2020. Ultracentrifuge clean-up of phage lysate for TEM. protocols.io https:// Copy   

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