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Authors: Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson
Summary: This protocol describes MesoScale Discovery (MSD) assays  for measurement of proinflammatory cytokines in supernatants collected from T cell dependent cytotoxicity (TDCC) cultures after BiTE® treatment.

Proper citation: Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson 2017. MSD 96-well human cytokine assay. protocols.io https://dx.doi.org/10.17504/protocols.io.hwzb7f6 Copy   

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Authors: Sam Alsford
Summary: Protocol obtained from the Alsford Lab at the London School of Hygiene & Tropical Medicine(https://blogs.lshtm.ac.uk/alsfordlab/protocols/bloodstream-form-culture/)

Proper citation: Sam Alsford 2018. Bloodstream-form culture. protocols.io https://dx.doi.org/10.17504/protocols.io.qhtdt6n Copy   

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Authors: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru
Group: Neurodegeneration Method Development Community
Summary: We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

Proper citation: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru 2019. Systemic AAV vectors for widespread and targeted gene delivery in rodents. protocols.io https://dx.doi.org/10.17504/protocols.io.84ahyse Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol can be used to dissociate adult human kidney “on ice” - maintaining authentic gene expression profiles. It was designed using a mix of Collagenases (Type 4, and A) which provide broad proteolytic activity, but preferentially cleave extracellular bonds, largely leaving cells intact. The total incubation time is 1 hour 20 minutes divided into two layers. At the end of the procedure, RBC lysis is performed. The total yield at the end of the procedure is ~1200 (non-RBC) cells released per mg tissue with 87% viability.In the digest mix, there is trypsin inhibitor from soybean which is designed to limit the activity of tryptic proteins in the collagenase mix which can damage the integrity of the cell. There is also 5 mM CaCl2, an activator of collagenase activity, in addition to DNAse - which chews up DNA released from dead cells, reducing cell clumping. The dissociation itself it carried out in two layers. The first layer is 30 minutes and includes trituration and shaking. After this layer, tissue clumps are settled for 1 min, and the supernatant containing released cells is removed and filtered using a 30 µM filter and rinsed with ice-cold PBS-BSA. This helps to preserve the integrity of released cells while continuing the digest clumps of undissociated cells. To the residual clumps, an additional 1 mL of enzyme mix is added and the digestion is continued for 50 additional minutes (1 hr 20 mins total time).

Proper citation: Andrew Potter 2018. Adult human kidney tissue cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q6cdzaw Copy   

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Authors:
Group: Metabolomics Protocols & Workflows, Translational Mass Spectrometry Research Group
Summary: Multiplex targeted proteomic assay to measure human proteins associated with inflammation in serum. A unique peptide is selected to represent a protein and is measured using multiple reaction monitoring (MRM) mass spectrometry.

Proper citation: 2020. MRM-LC-MS/MS Assay for inflammatrory associated proteins in serum. protocols.io https://dx.doi.org/10.17504/protocols.io.bm4vk8w6 Copy   

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Authors: Kayoko Morio, Yasuko Kurata, Nobuko Kawaguchi-Sakita, Akihiro Shiroshita, Yuki Kataoka

Proper citation: Kayoko Morio, Yasuko Kurata, Nobuko Kawaguchi-Sakita, Akihiro Shiroshita, Yuki Kataoka 2020. Efficacy of metformin in patients with breast cancer receiving chemotherapy or endocrine therapy：Systematic review and meta-analysis (protocol). protocols.io https://dx.doi.org/10.17504/protocols.io.bmzak72e Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Sandwich ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.98xh9xn Copy   

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Authors: Jeffrey Ardell
Group: SPARC
Summary: Yorshire pigs are anthesized for ICN/ neuromodulation recodings. Montior hemodynics A1mid-sternotomy

Proper citation: Jeffrey Ardell 2019. Pig ICN recording. protocols.io https://dx.doi.org/10.17504/protocols.io.2jugcnw Copy   

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Authors: Kelsey Knight
Group: BioLegend
Summary: Immunoprecipitation is a procedure by which proteins or peptides that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics. Immunoprecipitation can also be used to “enrich” a protein population prior to Western Blotting. For example, one can perform immunoprecipitation with a pan-specific antibody against a protein of interest followed by Western blotting with a modification-specific antibody (such as a phospho-specific antibody or an acetylation-specific antibody).

Proper citation: Kelsey Knight 2018. Immunoprecipitation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tkmeku6 Copy   

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Authors: Andreea S
Group: iGEM Groningen 2020
Summary: The potato root secretes root exudates, a mixture of organic molecules out of which solanine is found in the highest concentration (range of ug/ml). Also, solanine is a molecule specifically found in the root exudate of potato plants, which makes it a good candidate for a dependecy molecule which will ensure that Bacillus mycoides doesn't escape the designated action area. Studies have shown that there are soil bacteria able to metabolize solanine as a carbon source. Unfortunately, we weren't able to find an already described solanine-inducing promoter. Because the natural habitat of B. mycoides is the potato rhizosphere, we have strong reasons to believe that if such a promoter may already be present in its genome. In order to find this promoter, we designed a CHIP-Seq experiment. B. mycoides will be incubated with a different range of solanine concentrations. The DNA will be extracted and the CHIP-Seq technique will be used to detect potential binding sites of solanine operators using monoclonal anti-solanine antibodies.

Proper citation: Andreea S 2020. Transcriptomics. protocols.io https://dx.doi.org/10.17504/protocols.io.bme9k3h6 Copy   

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Authors: Tomasz Suchan
Group: Molecular Biogeography Group
Summary: Because of the influence of salts on enzymatic reactions, Qiagen extractions can be purified before making RAD tags. First, it is necessary to perform a chloropane extraction (phenol / chloroform 50/50) by adding SDS to the aqueous phase (final conc. 0.1%), this eliminates contaminants introduced by the matrix of the column. Then, perform an ethanol precipitation followed by a wash with 70% ethanol. This is to eliminate the salt that was used to bind the nucleic acids to the column and also to remove EDTA (inhibitor!) which is 0.5 mM in the buffer AE. Eluting with water is nonsense as the pH is not optimal and thus yield is poor and the DNA is not buffered and there is always too much salt.

Proper citation: Tomasz Suchan 2020. Phenol-chloroform DNA purification. protocols.io https://dx.doi.org/10.17504/protocols.io.re6d3he Copy   

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Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: Hurwitz Lab, Metafunc Course 2017
Summary: Set up your Unix PATH so you can find important tools on the HPC for the class.

Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2017. Setting $PATH on UA HPC. protocols.io https://dx.doi.org/10.17504/protocols.io.jh8cj9w Copy   

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Authors: Worapaka Manosroi

Proper citation: Worapaka Manosroi 2019. Basal cortisol. protocols.io https://dx.doi.org/10.17504/protocols.io.7iphkdn Copy   

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Authors: Anand Patel, Rachel T Noble, Joshua A Steele, Michael S Schwalbach, Ian Hewson, Jed A Fuhrman
Group: VERVE Net, Fuhrman Lab
Summary: The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously. The method is from:Patel A, Noble RT, Steele JA, Schwalbach MS, Hewson I, Fuhrman JA.Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I. Nat Protoc. 2007;2(2):269-76.Please see the full manuscript for more details.

Proper citation: Anand Patel, Rachel T Noble, Joshua A Steele, Michael S Schwalbach, Ian Hewson, Jed A Fuhrman 2018. Virus and bacteria counts by epifluorescence microscopy with SYBR Green. protocols.io https://dx.doi.org/10.17504/protocols.io.r2dd8a6 Copy   

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Authors: Lorane Texari, Carlos Guzman, Sven Heinz
Summary: Understanding the precise regulation of transcriptional programs in human health and disease requires the accurate identification and characterization of genomic regulatory networks. Next-generation sequencing (NGS) technologies are powerful, and widely applied tools to map the in vivo genome-wide location of transcription factors (TFs), histone modifications, chromatin accessibility, and nascent transcription that make up these regulatory networks. While chromatin immunoprecipitation followed by sequencing (ChIP-seq) is one of the oldest, and most-utilized experimental techniques to study the location and abundance of TFs, experiments still frequently require optimization to reproducibly yield good data with high signal-to-noise ratios due to the massive variability between possible antibody-antigen combinations and commercial reagents. .justify:after { content: ""; display:inline-block; width: 100%; } To overcome these obstacles, we systematically carried out well over 500 ChIP-seq experiments designed to test every aspect of typical ChIP-seq experiments and developed rev-ChIP, a novel ChIP-seq method that is optimized for scalability, robustness, low-input, speed, cost efficiency and data quality. We find that rev-ChIP can be scaled to work for cell numbers ranging from millions to under a thousand, and from a single sample to 500 samples a week in a non-automated fashion with minimal hands-on time. Additionally, rev-ChIP has been tested on a variety of sample types ranging from cell lines to sorted primary cells and solid tissues. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Lorane Texari, Carlos Guzman, Sven Heinz 2018. rev-ChIP. protocols.io https://dx.doi.org/10.17504/protocols.io.vp5e5q6 Copy   

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Authors: Maira Rivera, Javiera Reyes, Paula Blazquez-Sanchez, Cesar A Ramirez-Sarmiento
Group: Reclone.org (The Reagent Collaboration Network)

Proper citation: Maira Rivera, Javiera Reyes, Paula Blazquez-Sanchez, Cesar A Ramirez-Sarmiento 2020. Recombinant expression and purification of codon-optimized M-MLV and Mashup. protocols.io https:// Copy   

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Authors: Beeke Wienert
Group: CornLab
Summary: Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site, but can also affect off-target sites. We develop a powerful assay for the unbiased identification of off-target sites that we term DISCOVER-seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double strand breaks. One such factor, MRE11, is recruited so precisely to a double stranded break that nuclease cut sites can be determined with single-base resolution. DISCOVER-seq is applicable to multiple types of Cas nucleases and provides an unprecedented molecular picture of events that precede repair of the affected sites. DISCOVER-seq furthermore detects off-targets in cellular models and tissues.

Proper citation: Beeke Wienert 2018. DISCOVER-Seq: MRE11 ChIP Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.uvuew6w Copy   

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Authors: Courtney Comrie

Proper citation: Courtney Comrie 2020. Drawing ROIs in ITK-Snap. protocols.io https://dx.doi.org/10.17504/protocols.io.8nvhve6 Copy   

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Authors: Stephen Floor
Group: Stephen Floor Lab
Summary: This protocol is for in vitro transcription of long RNAs off plasmid or PCR product templates. It is assumed that the template has been gel purified, is the right size, has a T7 promoter and a 3' polyA tail (typically A60). Following this protocol the RNA will be ready for transfection into mammalian cells or in vitro translation. Significant amounts of template are necessary: at least 1ug of template per 100ul reaction; ideally closer to 5ug of template, especially for very long templates. Use RNase sensitive protocols and reagents for all steps of this procedure. The basic protocol is: - PCR amplification of the template- in vitro transcription- capping and 2'-O-Methylation - quality control and optional purification Based on protocols from Kaihong Zhou (Doudna lab) and RNA: A Laboratory Manual (Rio, Ares, Hannon, Nilsen).

Proper citation: Stephen Floor 2019. In vitro transcription, capping, and 2'-O methylation of long RNAs. protocols.io https://dx.doi.org/10.17504/protocols.io.4pwgvpe Copy   

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Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol describes confocal microscopy and image analysis procedures for characterizing neuronal cell bodies and their associated synaptic boutons in thick (50 µm) cryosections. The protocol has been applied to rat pelvic ganglia, where neuronal cell bodies have been identified using immunohistochemical markers of specific neuron populations and/or fluorescent retrograde tracer.

Proper citation: Janet Keast, Peregrine Osborne 2020. Confocal microscopy and characterization of synaptic boutons associated with ganglion neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bakdics6 Copy   

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