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Authors: Kenneth Schackart
Group: Yoon Lab
Summary: How to thaw cells from the liquid nitrogen storage and seed into a tissue culture flask

Proper citation: Kenneth Schackart 2019. Thawing and Seeding Frozen Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.xkmfku6 Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: List of published work using this procedure:

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PBMC- 03 - TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit”. protocols.io https://dx.doi.org/10.17504/protocols.io.bjabkian Copy   

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Authors: Anna Behle
Group: Axmann Lab
Summary: Isolation of plasmid DNA from E. coli using the alkaline lysis method modified from Birnboim et al., 1979. This protocol is suitable for fast, cheap recovery of large amounts of plasmid, e.g. for cloning purposes or restriction analysis. However, the purity of plasmid is insufficient for sequencing.

Proper citation: Anna Behle 2017. Isolation of plasmid DNA from E. coli (Alkaline lysis method). protocols.io https://dx.doi.org/10.17504/protocols.io.gtbbwin Copy   

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Authors: Thomas Skipper, Daniel Bondeson, Brenton Paolella, Francisca Vazquez
Group: Cancer Dependency Map Target Validation

Proper citation: Thomas Skipper, Daniel Bondeson, Brenton Paolella, Francisca Vazquez 2020. In vivo Nanopool: Pooled sgRNA competition assays to evaluate in vivo cancer dependencies. protocols.io https:// Copy   

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Authors: Xiaoyun Lei, Shi Huang

Proper citation: Xiaoyun Lei, Shi Huang 2017. Enrichment of minor allele of SNPs and genetic prediction of type 2 diabetes risk in British population. protocols.io https://dx.doi.org/10.17504/protocols.io.j7icrke Copy   

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Authors: Li Kang
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Mice with catheters implanted in the jugular vein (infusions) and carotid artery (sampling) are used for this procedure (V3002). The hyperinsulinemic, euglycemic clamp or “insulin clamp” has been used in a variety of species to assess insulin action. In an insulin clamp, the rate of glucose infused to maintain euglycemia is an index of whole body insulin sensitivity. Isotopes can be used during an insulin clamp to distinguish between insulin’s effects on endogenous glucose production and glucose utilization, or to examine insulin’s effects on specific tissues and metabolic pathways. The insulin infusion rate used in a study depends on a number of factors such as whether the experiment requires a sensitive analysis of endogenous glucose production, or whether the mouse is known to be unusually insulin resistant (e.g. ob/ob). Finally the insulin clamp technique, especially when isotopes are involved, is most quantitative under steady state conditions. Therefore, clamps should be of a duration (~2 h) that is sufficient for the actions of insulin to obtain steady state. The section that follows describes, a) the technical procedures used on the day of a clamp experiment; b) preparation of insulin and tracer infusates; and c) analyses of plasma and tissue radioactivity. Familiarity with these procedures combined with laboratory demonstration provides the investigator with the necessary background to clamp the conscious mouse.

Proper citation: Li Kang 2019. Vandy - Hyperinsulinemic-euglycemic Clamp. protocols.io https://dx.doi.org/10.17504/protocols.io.yyafxse Copy   

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Authors: Priota Islam
Group: Behavioural Genomics

Proper citation: Priota Islam 2020. Microbiome Assay with 96WP Updated April 2020. protocols.io https://dx.doi.org/10.17504/protocols.io.bfrqjm5w 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: After the cultivation period, cells were collected by centrifugation at 3500 × g for 30 min at 4 °C and resuspended in 4 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole and 0.05% Tween 20 at a pH of 7). The mixture was sonicated with 4 × 40 sec pulses followed by 20 sec rest between cycles at 4 °C. The crude extracts were then centrifuged at 9000 × g for 30 min at 4 °C, and the resulting supernatant was heat-treated in a hot water bath at 90 °C for 15 min. Insoluble material was separated by centrifugation at 13000 × g for 10 min, and the supernatant, containing the enzyme, was analyzed on a 12% SDS–polyacrylamide. The purified elution fractions were dialysed overnight in a 50 mM potassium-phosphate buffer (pH=7) at 4 °C.

Proper citation: Hossein Tarrahimofrad, Amir Meimandipour, Sareh Arjmand, Mohammadtaghi Beigi Nassiri, Ehsan Jahangirian, Hossein Tavana, Javad Zamani, Somayyeh Rahimnahal, Saeed Aminzadeh 2020. Single-step purification by heat shock. protocols.io https://dx.doi.org/10.17504/protocols.io.bgbsjsne Copy   

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Authors: Caroline Grabbe, Ingrid Dacklin, Behzad Khoshnood

Proper citation: Caroline Grabbe, Ingrid Dacklin, Behzad Khoshnood 2017. Flag M2 immunoprecipitation and Western Blot in Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.jivcke6 Copy   

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Authors: Jon Eilers
Summary: This protocol is for anesthetizing sea cucumbers. After soaking the sea cucumber in the menthol/ethanol/saltwaer solution for 30-60 minutes the sea cucumber should be flacid and relatively unresponsive to poking. No sea cucumber has eviscerated during surgery after being anesthetized although they do contract and appear like they are trying. The anesthestia wears off quickly after being placed back in salt water. This protocol is based on the method for anesthetizing Apostichopus japonicus as published in the citation below

Proper citation: Jon Eilers 2019. Anesthetizing Sea Cucumbers. protocols.io https://dx.doi.org/10.17504/protocols.io.5nyg5fw Copy   

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Authors: New England Biolabs

Proper citation: New England Biolabs 2020. Protein Synthesis Reaction using PURExpress (E6800). protocols.io https:// Copy   

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Authors: Leo Blondel
Summary: for 500ml (2x250ml)

Proper citation: Leo Blondel 2016. Growing and harvesting Electro Competent E-coli Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.gc7bszn Copy   

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Authors: Eric Gann
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)

Proper citation: Eric Gann 2017. Transformation of Aureococcus anophagefferens by Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.g2vbye6 Copy   

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Authors: Bing Yang

Proper citation: Bing Yang 2019. EpiTect Plus Bisulfite Conversion. protocols.io https://dx.doi.org/10.17504/protocols.io.3bfgijn Copy   

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Authors: Angel Justiz-Vaillant
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. Th9, Th2, Th17, mast cells, NKT cells, and regulatory T cells produce interleukin-9. It enhances T-cell survival, mast cell activation and synergy with erythropoietin.[1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. Copy of ELISA for quantification of IL-7 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj28kqhw Copy   

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Authors: Benjamin S. Glicksberg, Roberto Mora, Stefano Rensi, Vivek A. Rudrapatna, Andrew M. Bishara, Elizabeth Gress, Rohit Vashisht, Michael Zobel, Benjamin Padilla, Benjamin Padilla, Atul J. Butte
Summary: Randomized control trials (RCTs) are the gold standard for clinical trials to evaluate effectiveness and safety of interventions. Conducting RCTs, however, is not always feasible, particularly for rare conditions. Real-world data (RWD), including clinical information from Electronic Health Records (EHR), are utilized for similar analyses in retrospective data. The United States Food and Drug Administration are increasingly receptive of accepting such analyses as evidence as long as the studies are designed and conducted robustly. EHR work suffers from a reproducibility crisis due to such factors such as hospital-specific biases, among others. Akin to preregistration of RCTs, devising, detailing, and publishing protocols of RWD before conducting analyses can enhance reproducibility and fidelity of results. In the current work, we release a detailed protocol to assess effectiveness and safety of a cryoablation intervention compared to epidural for pain control in adolescent patients undergoing the Nuss procedure in the retrospective EHRs of a major hospital system.

Proper citation: Benjamin S. Glicksberg, Roberto Mora, Stefano Rensi, Vivek A. Rudrapatna, Andrew M. Bishara, Elizabeth Gress, Rohit Vashisht, Michael Zobel, Benjamin Padilla, Benjamin Padilla, Atul J. Butte 2020. Assessment of safety and effectiveness for pediatric patients of cryoanalgesia compared to epidural for the Nuss procedure in real-world data from Electronic Health Records: a protocol and statistical analysis plan. protocols.io https://dx.doi.org/10.17504/protocols.io.bbpaimie Copy   

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Authors: Lenny Teytelman, Liz Silva, Joanne Kamens, Lex Flagel
Group: Academic Career Forum
Summary: [Please use the commenting functionality to suggest edits and additional resources. Also, please feel free to use the “copy/fork” button to make a different version of this for your field/institute/students.]In the 1960s, 60% of biomedical PhDs went on to tenure track faculty positions. Today, that fraction is estimated to be 10%-15% of graduate students. Similar trends exist across the biological sciences. This shift means that over the years, many of the students and postdocs in your lab will also explore different career options, and as a good mentor, being able to support them is important, no matter which career track they choose.One of the challenges for professors is that they are deeply familiar with the academic track, but are often at a loss in recommending resources and steps for the trainees who are not 100% certain that they will become faculty as well.This guide is meant to be a starting point. It can be easily shared with the trainees and offers simple tips for career exploration. The goal is not to steer trainees away from academia, but to help them figure out whether academia is the right fit, and if not, how they can explore the many different options that are available to them to find a rewarding and satisfying path.(Image: Flazingo Photos, "Newspaper - Careers", CC BY-SA 2.0)

Proper citation: Lenny Teytelman, Liz Silva, Joanne Kamens, Lex Flagel 2020. Guide for Supporting Diverse Career Exploration of Trainees. protocols.io https://dx.doi.org/10.17504/protocols.io.8mwhu7e Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: 3, 3´ Diaminobenzidine (DAB) is a commonly used chromogen for histological staining. In the presence of peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol and xylene. Peroxidase catalyzes hydrogen peroxide oxidation of substrate DAB. This SOP provides instructions to prepare four different formulations of DAB. DAB for Immunohistochemical staining (IHC), DAB for Acetylcholinesterase (AChE) Staining, Nickel Enchanced DAB for Biocytin staining, and DAB for Biocytin staining.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 Solutions. protocols.io https://dx.doi.org/10.17504/protocols.io.bew7jfhn Copy   

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Authors: Bonnie Hurwitz
Group: Hurwitz Lab, ABE487_HMP, ABE487_EMP
Summary: In a study comparing obese versus lean twins, Turnbaugh and colleagues found that there were "shared microbial genes among sampled individuals, comprising an extensive, identifiable 'core microbiome' at the gene, rather than at the organismal lineage level".  This means that despite having differences in bacterial composition, the "core" functional repertoire of microbiome remains the same in obese versus lean twins.  In this tutorial, we are going to explore these functional differences in two lean twins (TS1 and TS2) and two obese twins (TS19 and TS20) from this study.   Turnbaugh, Peter J., et al. "A core gut microbiome in obese and lean twins."nature 457.7228 (2009): 480-484.

Proper citation: Bonnie Hurwitz 2015. Finding the core vs flexible microbiome. protocols.io https://dx.doi.org/10.17504/protocols.io.d2y8fv Copy   

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Authors: Luciano Martelotto
Group: Human Cell Atlas Method Development Community, Single Cell Core, Harvard Medical School
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.bsxhnfj6 Copy   

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