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Authors: Margot K Brandt, Alper Gokden, Marcello Ziosi, Tuuli Lappalainen
Summary: Polyclonal allelic expression assay for detecting the regulatory effects of genetic variants within transcripts using CRISPR/Cas9 genome editing and amplicon sequencing.

Proper citation: Margot K Brandt, Alper Gokden, Marcello Ziosi, Tuuli Lappalainen 2019. Polyclonal Allelic Expression Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.2jigcke Copy   

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Authors: Michael Eadon, Daria Barwinska, Ying-Hua Cheng, Michael J. Ferkowicz, Samir V. Parikh, Brad H. Rovin, John P. Shapiro, Pierre C. Dagher, Tarek M. El-Achkar
Group: KPMP
Summary: Recent advances in multi-scale interrogation of human tissue, including advanced imaging techniques and the powerful application of large dataset “omics”, have displayed significant promise toward identifying new and specific therapeutic targets, predicting disease progression, and individualizing treatment in participants with acute and chronic kidney disease. Our site, formed by the alliance of Indiana University and Ohio State University, will implement an unbiased tissue interrogation workflow for KPMP human kidney biopsies that integrates largescale 3D tissue imaging for quantitative supervised and unsupervised analysis/cytometry with sub-segmental “omics” data on the same kidney biopsy specimen. The sub-segmental “omics” pipeline will use fluorescence based Laser MicroDissection (LMD) to isolate specific nephron segments and interstitial/other targeted areas, for downstream analysis with transcriptomics and proteomics. The omics analysis will be eventually expanded to include bulk epigenetics. Acting harmoniously with other KPMP sites, our interrogation techniques are expected to facilitate back-mapping of key molecular pathways to the biopsy, which can subsequently identify foci of injury and/or regeneration that can undergo targeted sampling to generate further enriched omics. Therefore, in addition to a significant contribution to the human kidney atlas, our approach will complement other interrogation techniques within KPMP by providing tissue context and increasing spatial resolution for molecular signatures that arise in heterogeneous areas during kidney disease.

Proper citation: Michael Eadon, Daria Barwinska, Ying-Hua Cheng, Michael J. Ferkowicz, Samir V. Parikh, Brad H. Rovin, John P. Shapiro, Pierre C. Dagher, Tarek M. El-Achkar 2020. Laser microdissection for regional transcriptomics and proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.bew6jfhe Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads and less antibody than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Selection Kits Column Protocol - 3. protocols.io https://dx.doi.org/10.17504/protocols.io.7tvhnn6 Copy   

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Authors: Shuangshuang Li

Proper citation: Shuangshuang Li 2017. Transwell migration assay. protocols.io https://dx.doi.org/10.17504/protocols.io.h4ib8ue Copy   

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Authors: Li Deng, Bonnie T. Poulos, Matthew B. Sullivan
Group: VERVE Net, Sullivan Lab
Summary: AbstractOcean viruses are abundant, ubiquitous, and play important roles in global biogeochemical cycles through mortality, horizontal gene transfer and manipulation of host metabolism. However, the ability to link viruses to their hosts in a high-throughput manner bottlenecks our ability to understand virus-host interactions in complex communities. Here, we present viral tagging (VT), a method that combines mixtures of isotope labeled host cells and fluorescent viruses with flow cytometry. In a single experiment, we can screen 107 uncultivated ocean viruses with a single strain of Synechococcus. These viruses can then be sequenced to quantitatively link objectivelydefined environmental viral populations, and their genomes, to their hosts.

Proper citation: Li Deng, Bonnie T. Poulos, Matthew B. Sullivan 2016. Obtaining targeted metagenomes from uncultivated environmental Synechococcus using flow cytometry-based viral tagging. protocols.io https://dx.doi.org/10.17504/protocols.io.c6zzf5 Copy   

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Authors: Tomasz Suchan
Group: Molecular Biogeography Group

Proper citation: Tomasz Suchan 2018. AMPure purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.sk7eczn Copy   

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Authors: Regina Hoo, Roser Vento-Tormo
Group: Vento-Tormo
Summary: This protocol is for enrichment of epithelial glands on endometrium; following the step from "Endometrium dissociation with collagenase".

Proper citation: Regina Hoo, Roser Vento-Tormo 2020. Endometrium dissociation with trypsin . protocols.io https://dx.doi.org/10.17504/protocols.io.72dhqa6 Copy   

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Authors: Ian Mackay, Judy Northill
Summary: This assay is a modification to the World Health Organization's influenza A H3 TaqMan documented in 'WHO information for molecular diagnosis of influenza virus - update 1' (see file below). New primers were added and the WHO primers discarded.

Proper citation: Ian Mackay, Judy Northill 2019. Influenza A H3 virus TaqMan assay. protocols.io https://dx.doi.org/10.17504/protocols.io.2qwgdxe Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocols is for PCR using Q5® High-Fidelity DNA Polymerase (M0491)

Proper citation: New England Biolabs 2015. PCR with Q5® Polymerase (M0491). protocols.io https://dx.doi.org/10.17504/protocols.io.cidua5 Copy   

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Authors: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin
Summary: The first procedure for implementing the ARF-AID system is to establish ARF-TIR1 progenitor cells, as shown in Figure 2A. All the plasmids for integrating ARF-TIR1 into the AAVS1 locus in human cells are available from Addgene. The choice of transfection method varies depending on the cell type; lipofectamine 3000 works efficiently for HEK293T cells.

Proper citation: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin 2020. Basic Protocol 1: Generation of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 progenitor cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bde4i3gw Copy   

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Authors: Kwangok P Nickel, Austin M. Maas, Randall Kimple
Summary: Cancer stem cells (CSCs) are a small subpopulation of tumor cells that are thought to be responsible for recurrence and metastasis of cancer due to their ability for self-renewal and differentiation into multiple cancer cell types. CSCs are also known to play a key role in the development of metastases. Cancer cells undergoing Epithelial to Mesenchymal Transition (EMT) have also been shown to attain CSC phenotypes and acquire invasive and migratory properties. Therefore, understanding the characteristics of CSCs may help to develop better treatment strategies. The sphere formation assay has been widely used to isolate CSCs using anchorage-independent sphere culture. CSCs can grow on ultra-low attachment plates that are coated with a layer to inhibit the attachment of cells. When cells are grown in serum-free and non-adherent conditions, CSCs can survive and clonally expand to form spheres, whereas differentiated tumor cells undergo apoptosis due to their anchorage dependence.

Proper citation: Kwangok P Nickel, Austin M. Maas, Randall Kimple 2020. Isolation of cancer stem cells by sphere formation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bdt8i6rw Copy   

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Authors: Chad Vezina

Proper citation: Chad Vezina 2020. Vezina Lab IHC Protocol. protocols.io https:// Copy   

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Authors: Joshua Welsh, Julia Kepley, Bryce Killingsworth, Tim Traynor, Jennifer Jones
Group: Translational Nanobiology Section
Summary: Protocol for ultrafiltration of cell culture conditioned media using Pall Jumbosep products and purification of EVs from conditioned media concentrate using Izon qEV-10 size exclusions columns.

Proper citation: Joshua Welsh, Julia Kepley, Bryce Killingsworth, Tim Traynor, Jennifer Jones 2020. Ultrafiltration and purification of conditioned media (Pall Jumbsosep and Izon qEV-10)  . protocols.io https://dx.doi.org/10.17504/protocols.io.beqnjdve Copy   

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Authors: Sierra Simpson, Olivier George
Group: George Lab

Proper citation: Sierra Simpson, Olivier George 2021. GNPS Untargeted Metabolomics Workflow . protocols.io https:// Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to generate accurate digital representations of neuron morphologies from a variety of brain regions and species. Each reconstruction captures the positions and thicknesses of the soma, dendrites and axon of a biocytin-filled cell within a slice of brain tissue. To generate the reconstruction we use an image stack containing ~200-700 serial 2D images that captures the full extent of the cell within the slice. We use the Vaa3D (Terafly) program with a Kazom’s Mozak user interface (Mozak for short) to visualize the 2D images in 3D. Once the stack is loaded in Mozak, our reconstruction is generated by placing nodes in 3D space. The placement of these nodes is dependent on the signal in the images. Our final output, an SWC text file (.swc format), contains many thousand rows. Each row contains a node ID, an x, y, z coordinate, radius value, neurite type, and parent node ID. After tracing is complete, we preform post processing to provide radius values, check for errors and consistency then the SWCs are uploaded into our Laboratory Information Management 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. 3D Reconstruction of Neurons in Vaa3D. protocols.io https://dx.doi.org/10.17504/protocols.io.bdppi5mn Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: This kit is designed for the isolation of untouched CD14+CD16- monocytes from peripheral blood mononuclear cells (PBMCs). Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc. Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Human CD14+ Monocytes Isolation Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7x9hpr6 Copy   

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Authors: IIDP-HIPP
Group: Integrated Islet Distribution Program and Human Islet Phenotyping Program
Summary: This Standard Operating Procedure (SOP) is based on the Vanderbilt Human Islet Phenotyping Program (HIPP) procedures for dispersing islets to single cell suspension and quantitatively assessing viability by Trypan Blue staining.This SOP defines the assay method used by the Human Islet Phenotyping Program (HIPP) for quantitative and qualitative determination of the Purified Human Pancreatic Islet product, post-shipment, manufactured for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP).

Proper citation: IIDP-HIPP 2020. Quantitative Assessment of Islet Viability upon Arrival to HIPP by Staining with Trypan Blue Dye. protocols.io https://dx.doi.org/10.17504/protocols.io.bp97mr9n Copy   

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Authors: Tobias Weise, Bettina Boettcher, Slavena Vylkova
Summary: Biofilm formation under shear flow conditions was monitored using the Bioflux1000 device (Fluxion Biosciences, Inc.). In short, Candida albicans overnight cultures were washed in pre-warmed RPMI medium. Cells were seeded for 2-5 sec from the outlet well into the channels of Bioflux1000 flow chambers, which were primed before with warm medium. The cells were allowed to adhere to the channels for 90 min without any flow, followed by removal of non-adherent cells by flowing fresh, pre-warmed RPMI medium for 5 sec. Shear flow was set for time series experiments over 24 h biofilm formation and images were captured every 20 min. Two channels were investigated in parallel having a 10 × magnification to allow a direct comparison between a mutant and a reference (wild-type) strain. Image capturing and stacks to movies was performed using the MetaMorph® Software (Molecular Devices).An ODE model reflecting the logistic growth as well as the lag phase was fitted to the individual experiments. Fitting was carried out by minimising a cost function (unweighted least-squares-based) using the Nelder-Mead algorithm. Growth rate time series generated from the fitted model were used to compare wild type and mutant regarding the maximum observed growth rates at their respective time points.All computations were performed using the programming language python (version 3.6.9) and the additional packages numpy (version 1.16.2), pandas (version 0.25.0), and scipy (version 1.3.1).

Proper citation: Tobias Weise, Bettina Boettcher, Slavena Vylkova 2020. Bioflux Analyses: Modelling. protocols.io https://dx.doi.org/10.17504/protocols.io.bb7sirne Copy   

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Authors: Sebastien Colin
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Tara Oceans
Summary: This protocol describes how to prepare, stain and mount biogenic silica particles (opaline from diatoms, Radiolaria, sponges spicules, phytolith...) for 3D imaging with fluorescent microscopy. The protocol is fast, convenient and inexpensive. Hence it could be a relevant alternative to X-ray computed tomography or photogrammetry.

Proper citation: Sebastien Colin 2018. Simple 3D imaging of biogenic silica structures by fluorescent microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.vd8e29w Copy   

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Authors: Eugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein
Summary: Detailed cell electroporation protocol from paper:Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots Eugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein

Proper citation: Eugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein 2017. Electroporation of COS-7 cells and functionalization of QDs. protocols.io https://dx.doi.org/10.17504/protocols.io.g2mbyc6 Copy   

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