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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. T Cell Activation with anti-CD3 Antibodies Protocol - Human. protocols.io https://dx.doi.org/10.17504/protocols.io.793hr8n Copy   

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Authors: Steven Burgess
Group: OpenPlant Project, Plantae
Summary: Salt solution used in preparation of minimal medium for Chlamydomonas reinhardtii.Source:Sueoka, N. (1960) Proc. Natl. Acad. Sci. USA 46, 83-91.http://www.chlamycollection.org/Sueoka.html

Proper citation: Steven Burgess 2016. Beijerinck's Solution. protocols.io https://dx.doi.org/10.17504/protocols.io.fdgbi3w Copy   

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Authors: Luciano Martelotto
Summary: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small sample sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al.This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies.The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other samples types.Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.

Proper citation: Luciano Martelotto 2020. Frankenstein’ protocol for nuclei isolation from FRESH and FROZEN tissue for snRNA-Seq (10x Genomics Platform). protocols.io https://dx.doi.org/10.17504/protocols.io.bq24mygw Copy   

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Authors: Sarah Urata, Blue Lake, Dinh Diep, Masato Hoshi, Sanjay Jain, Kun Zhang
Group: KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: 10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. Here we present a modified version of the published 10X protocol [1] that we have adapted for the processing of adult human kidney nuclei.References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2.

Proper citation: Sarah Urata, Blue Lake, Dinh Diep, Masato Hoshi, Sanjay Jain, Kun Zhang 2019. 10X Genomics Single-Nucleus RNA-Sequencing for Transcriptomic Profiling of Adult Human Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.86khzcw Copy   

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Authors: Ben Sutherland, Claire Rycroft, Kristina Miller
Summary: This protocol comes with no guarantees, and is primarily used for the authors’ purposes. This protocol is an adaptation from the work in the following publications:Mascher M, Wu S, Amand PS, Stein N, Poland J (2013) Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley. PLoS ONE 8(10): e76925. https://doi.org/10.1371/journal.pone.0076925Recknagel, H.,Jacobs, A.,Herzyk, P., &Elmer, K. R.(2015).Double‐digest RAD sequencing using ion proton semiconductor platform (ddRADseq‐ion) with nonmodel organisms.Molecular Ecology Resources,15,1316–1329.https://doi.org/10.1111/1755-0998.12406

Proper citation: Ben Sutherland, Claire Rycroft, Kristina Miller 2019. Nsil1-Msp1 RAD-digest Detailed Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.w95fh86 Copy   

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Authors: Alexandra Ehl, David Frommholz, Nadine Stefanczyk
Summary: Purification Guide for the Isolation of Antibodies with ChroPack Columns by DALEX Biotech.With ChroPack Protein A columns you can quickly isolate monoclonal and polyclonal antibodies from various sample materials. Antibodies bind with high specificity to the recombinant protein A whose BSA binding site has been removed. The ChroPack columns can be operated with peristaltic pumps and common FPLC systems. The Luer-Lock connections make changing the column particularly easy.One-time autoclavable with DALEX stabilizer for Protein A.Each ml bed volume has a binding capacity of > 10 mg (tested with polyclonal human Ig, the binding capacity varies according to species and clones).Up to 98 % purity in a single purification step.No swelling/shrinking of the matrix in aqueous buffers.High thermal stability, up to 15 minutes at 80 °C in aqueous buffers with neutral pH.pH stability: short-term pH 2 - 8, long-term pH 3 - 8.Suitable for all common low-pressure systems.

Proper citation: Alexandra Ehl, David Frommholz, Nadine Stefanczyk 2019. ChroPack - Protein A. protocols.io https://dx.doi.org/10.17504/protocols.io.72mhqc6 Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Phenotyping of Rodents for the Presence of Diabetic Neuropathy In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.Diabetic Complication:

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Basic Breeding to Harvest. protocols.io https://dx.doi.org/10.17504/protocols.io.3jngkme Copy   

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Authors: Uriel Barboza Perez, Ekaterina Krasnopeeva, Jerko Rosko, Teuta Pilizota
Summary: Preparation of our standard tunnel-slide, used for BFM speed measurements and experiments involving rapid media exchange.

Proper citation: Uriel Barboza Perez, Ekaterina Krasnopeeva, Jerko Rosko, Teuta Pilizota 2021. Tunnel Slide Preparation for Motor Speed Recordings . protocols.io https://dx.doi.org/10.17504/protocols.io.bcjdiui6 Copy   

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Authors: David Frommholz, Alexandra Ehl, Nadine Stefanczyk
Summary: Purification Guide for the Isolation of Antibodies with ChroPlate Filtration Plates by DALEX Biotech.Easy and quick high throughput antibody purification from various sources and species.Each well of the ChroPlate has a binding capacity of > 1 mg (tested with human polyclonal Ig, binding varies between species and clones).The proprietary resin does not shrink or swell in aqueous buffers.High pressure stability.pH stability short term 2 - 8, long term 3 - 8.Excellent thermal stability up to 15 minutes at 80 °C in aqueous buffers at neutral pH.Can be dried for long term storage (80 °C for > 2 h).

Proper citation: David Frommholz, Alexandra Ehl, Nadine Stefanczyk 2018. ChroPlate - ProteinA/G. protocols.io https://dx.doi.org/10.17504/protocols.io.uzqex5w Copy   

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Authors: Pedro Coelho N Diogenes, Aline Naiara Azevedo da Silva, Fausto Pierdoná Guzen, Marco Aurelio de Moura Freire, José Rodolfo Lopes de Paiva Cavalcanti

Proper citation: Pedro Coelho N Diogenes, Aline Naiara Azevedo da Silva, Fausto Pierdoná Guzen, Marco Aurelio de Moura Freire, José Rodolfo Lopes de Paiva Cavalcanti 2019. Upper limb superficial venous percussion as a sign of anatomical location and venous permeability. protocols.io https://dx.doi.org/10.17504/protocols.io.4s7gwhn Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II FS DNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time.Note: The Ultra II FS Kit is not compatible with bisulfite conversion workflows.

Proper citation: New England Biolabs 2021. E7805 NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® Protocol to use with Inputs ≥ 100 ng (Chapter 2). protocols.io https://dx.doi.org/10.17504/protocols.io.nwxdffn Copy   

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Authors: Fan Zhang, Jessica Weckhorst, Adrien Assie, Anastasia Khodakova, Mario Loeza Cabrera, Daniela Vidal, Christopher Ayoub, Buck Samuel
Summary: The gut microbiome is an important driver of host physiology and development. Altered abundance or membership of this microbe community can influence host health and disease progression, including the determination of host lifespan and healthspan. Here, we describe a robust pipeline to measure the microbiome abundance and composition in the C. elegans gut that can be applied to examine the role of the microbiome on host aging processes.

Proper citation: Fan Zhang, Jessica Weckhorst, Adrien Assie, Anastasia Khodakova, Mario Loeza Cabrera, Daniela Vidal, Christopher Ayoub, Buck Samuel 2020. High-throughput assessment of changes in the Caenorhabditis elegans gut microbiome. protocols.io https://dx.doi.org/10.17504/protocols.io.rtzd6p6 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to attain 10% Triton X-100 which is utilized in several Immunohistochemistry (IHC) reagents to reduce the surface tension of the reagents and to make the cell membranes of the tissue more permeable.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. 10% Triton X-100. protocols.io https://dx.doi.org/10.17504/protocols.io.bezhjf36 Copy   

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Authors: Kun-Han Lu, Zhongming Liu, Jaiyue Cao
Group: SPARC
Summary: Briefly, a gadolinium‐based contrast agent was mixed with the animal’s meal in order for chyme to appear “bright” in MRI scans, thereby delineating the gastric and intestinal volume. A multi‐slice MRI sequence was used to scan the GI volume with high spatial resolution, and a similar sequence with a smaller spatial coverage was used to scan antral contractions with high temporal resolution. Measurements of gastric functions and physiology included the overall change in GI volume, gastric emptying, forestomach volume, corpus volume, antral volume, antral contraction frequency, antral peristaltic wave velocity, antral contraction amplitude, pyloric opening size, intestinal filling and, indirectly, absorption.

Proper citation: Kun-Han Lu, Zhongming Liu, Jaiyue Cao 2019. Contrast-enhanced magnetic resonance imaging of gastric emptying and motility in rats. protocols.io https://dx.doi.org/10.17504/protocols.io.wvxfe7n Copy   

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Authors:
Group: UCSC BME 22L

Proper citation: 2020. Protocol 1: PCR. protocols.io https:// Copy   

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Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: Invitrogen’s patented Syto dyes are cell-permeant cyanine dyes that bind to nucleic acids. Several Syto dyes are available with varying cell permeability, fluorescence enhancement upon binding to nucleic acids, excitation and emission spectra, and nucleic acid selectivity and binding affinity. The Syto 60 stain has absorption and fluorescence emission maxima of 652/678 nm. Nucleic acids stained with the Syto 60 stain can be detected and quantified on the Odyssey® Infrared and Odyssey Fc Imaging Systems using the 700 nm channel. In the procedures outlined, the Syto 60 dye was used to stain serial dilutions of a 1 kb DNA ladder and a 50 bp DNA ladder (New England Biolabs, P/N N3232 and N3236, respectively). Three methods are presented for staining of DNA in this technical note. The Syto 60 stain can be included in the DNA sample for detection using an Odyssey Imaging system.The Syto 60 stain can also be combined with ethidium bromide (EtBr) and included in the DNA sample for visualization on an Odyssey Imaging System and on a UV transilluminator; or the Syto 60 stain can be diluted and used alone as a post-electrophoresis gel stain. For additional information, see Imaging Nucleic Acid Gels on the Odyssey Fc Imager.

Proper citation: LI-COR Biosciences 2018. Syto® 60 Staining of Nucleic Acids in Gels. protocols.io https://dx.doi.org/10.17504/protocols.io.gvnbw5e Copy   

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Authors: Alexander I. Culley, Curtis A. Suttle, and Grieg F. Steward
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: The diversity of ribonucleic acid (RNA) viruses in the ocean and the ongoing isolation and characterization of RNA viruses that infect important primary producers suggests that RNA viruses are active members of the marine microbial assemblage. At this point, little is known about the composition, dynamics, and ecology of the RNA virioplankton. In this collection, we describe two methods to assess RNA virus diversity from seawater.

Proper citation: Alexander I. Culley, Curtis A. Suttle, and Grieg F. Steward 2015. Characterization of the diversity of marine RNA viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.dxr7m5 Copy   

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Authors: Geoff Baldwin, Traci Haddock-Angelli, Jacob Beal, Ari Dwijayanti, Marko Storch, Natalie Farny, Richard Tennant, Paul Rutten
Group: iGEM Measurement
Summary: Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve. Although distribution of a known concentration of GFP protein would be an ideal way to standardize the amount of GFP fluorescence in our ​E. coli​ cells, the stability of the protein and the high cost of its purification are problematic. We therefore use the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).You will prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in your plate reader. By measuring these in your plate reader, you will generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration. Before beginning this protocol, ensure that you are familiar with the GFP settings and measurement modes of your instrument. You will need to know what filters your instrument has for measuring GFP, including information about the bandpass width (530 nm / 30 nm bandpass, 25-30 nm width is recommended), excitation (485 nm is recommended) and emission (520-530 nm is recommended) of this filter.Note: The iGEM Particle Standard Curve with Microspheres calibration method is a pre-requisite for carrying out this protocol. You will need data from that calibration to analyse the results of this protocol.

Proper citation: Geoff Baldwin, Traci Haddock-Angelli, Jacob Beal, Ari Dwijayanti, Marko Storch, Natalie Farny, Richard Tennant, Paul Rutten 2019. Calibration Protocol - Fluorescence Standard Curve with Fluorescein. protocols.io https://dx.doi.org/10.17504/protocols.io.5n6g5he Copy   

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Authors: Francesca Lake
Summary: A collection of protocols from BioTechniques

Proper citation: Francesca Lake 2019. BioTechniques. protocols.io https://dx.doi.org/10.17504/protocols.io.936h8re Copy   

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Authors: Sarah Giuliani
Group: Coleman Lab
Summary: Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.8 Hourly Timepoints: 0, 2, 4, 6, 8, 10, 12, 14Sampling is to determine total cell concentration by flow cytometry.

Proper citation: Sarah Giuliani 2016. Sampling for Flow Cytomtery (FCM) Cell Quantification: Large-scale One-step Phage Infection of Cyanobacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.fh6bj9e Copy   

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