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  • Publisher Correction: Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy
    Nat. Protoc. (IF 10.419) Pub Date : 2020-12-02
    Erin E. Diel; Jeff W. Lichtman; Douglas S. Richardson

    A Correction to this paper has been published: https://doi.org/10.1038/s41596-020-0360-2.

  • 3D active stabilization for single-molecule imaging
    Nat. Protoc. (IF 10.419) Pub Date : 2020-12-02
    Simao Coelho; Jongho Baek; James Walsh; J. Justin Gooding; Katharina Gaus

    A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we

  • Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes
    Nat. Protoc. (IF 10.419) Pub Date : 2020-12-02
    Byungil Kim; Sarah Arcos; Katherine Rothamel; Manuel Ascano

    The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA–host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled

  • Nondestructive production of exosomes loaded with ultrathin palladium nanosheets for targeted bio-orthogonal catalysis
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-27
    Victor Sebastian; María Sancho‐Albero; Manuel Arruebo; Ana M. Pérez‐López; Belén Rubio‐Ruiz; Pilar Martin‐Duque; Asier Unciti‐Broceta; Jesús Santamaría

    The use of exosomes as selective delivery vehicles of therapeutic agents, such as drugs or hyperthermia-capable nanoparticles, is being intensely investigated on account of their preferential tropism toward their parental cells. However, the methods used to introduce a therapeutic load inside exosomes often involve disruption of their membrane, which may jeopardize their targeting capabilities, attributed

  • Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-27
    Delilah Hendriks; Benedetta Artegiani; Huili Hu; Susana Chuva de Sousa Lopes; Hans Clevers

    The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that

  • Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-27
    Hendrik A. Messal; Jorge Almagro; May Zaw Thin; Antonio Tedeschi; Alessandro Ciccarelli; Laura Blackie; Kurt I. Anderson; Irene Miguel-Aliaga; Jacco van Rheenen; Axel Behrens

    Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the 3D appreciation of cell and protein localization in their native organ environment. However, the sample preparations for such imaging are often onerous, and their capability for antigen detection is limited. Here, we describe FLASH (fast light-microscopic analysis of antibody-stained

  • Doubling the resolution of a confocal spinning-disk microscope using image scanning microscopy
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-27
    Shun Qin; Sebastian Isbaner; Ingo Gregor; Jörg Enderlein

    Fluorescence microscopy has become an indispensable tool for cell biology. Recently, super-resolution methods have been developed to overcome the diffraction limit of light and have shown living cells in unprecedented detail. Often, these methods come at a high cost and with complexity in terms of instrumentation and sample preparation, thus calling for the development of low-cost, more accessible

  • Radiosynthesis of [ 18 F]SiFA lin- TATE for clinical neuroendocrine tumor positron emission tomography
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-23
    Simon Lindner; Carmen Wängler; Justin J. Bailey; Klaus Jurkschat; Peter Bartenstein; Björn Wängler; Ralf Schirrmacher

    Here, we describe an extension of our silicon fluoride acceptor (SiFA) protocol for 18F-labeling of peptides that addresses challenges associated with preparing a clinical-grade (Tyr3)-octreotate (TATE) tracer for diagnosis of neuroendocrine tumors (NETs). After several iterations of protocol optimization (e.g., finding the optimal pH at which the isotopic exchange (IE) reaction produces high radiochemical

  • Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-23
    Hisashi Miura; Saori Takahashi; Takahiro Shibata; Koji Nagao; Chikashi Obuse; Katsuzumi Okumura; Masato Ogata; Ichiro Hiratani; Shin-ichiro Takebayashi

    Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application

  • Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-18
    Joel I. Perez-Perri; Marko Noerenberg; Wael Kamel; Caroline E. Lenz; Shabaz Mohammed; Matthias W. Hentze; Alfredo Castello

    Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues

  • Phage-assisted continuous and non-continuous evolution
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-16
    Shannon M. Miller; Tina Wang; David R. Liu

    Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher

  • An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-16
    Sona Vodenkova; Amaya Azqueta; Andrew Collins; Maria Dusinska; Isabel Gaivão; Peter Møller; Alena Opattova; Pavel Vodicka; Roger W. L. Godschalk; Sabine A. S. Langie

    This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids (‘naked’ supercoiled DNA) containing specifically induced DNA lesions (e.g

  • Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-09
    Xiaoran Roger Liu; Don L. Rempel; Michael L. Gross

    The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution

  • Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-09
    Fadi Jacob; Guo-li Ming; Hongjun Song

    Glioblastoma tumors exhibit extensive inter- and intratumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Most in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe

  • High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-02
    Virginia De Cesare; Jennifer Moran; Ryan Traynor; Axel Knebel; Maria Stella Ritorto; Matthias Trost; Hilary McLauchlan; C. James Hastie; Paul Davies

    Deubiquitylating enzymes (DUBs) play a vital role in the ubiquitin pathway by editing or removing ubiquitin from their substrate. As breakthroughs within the ubiquitin field continue to highlight the potential of deubiquitylating enzymes as drug targets, there is increasing demand for versatile high-throughput (HT) tools for the identification of potent and selective DUB modulators. Here we present

  • Proximity labeling in mammalian cells with TurboID and split-TurboID
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-02
    Kelvin F. Cho; Tess C. Branon; Namrata D. Udeshi; Samuel A. Myers; Steven A. Carr; Alice Y. Ting

    This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein–protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially

  • Genome-wide detection of DNA double-strand breaks by in-suspension BLISS
    Nat. Protoc. (IF 10.419) Pub Date : 2020-11-02
    Britta A. M. Bouwman; Federico Agostini; Silvano Garnerone; Giuseppe Petrosino; Henrike J. Gothe; Sergi Sayols; Andreas E. Moor; Shalev Itzkovitz; Magda Bienko; Vassilis Roukos; Nicola Crosetto

    sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after

  • Author Correction: lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-30
    M. Grace Gordon; Fumitaka Inoue; Beth Martin; Max Schubach; Vikram Agarwal; Sean Whalen; Shiyun Feng; Jingjing Zhao; Tal Ashuach; Ryan Ziffra; Anat Kreimer; Ilias Georgakopoulos-Soares; Nir Yosef; Chun Jimmie Ye; Katherine S. Pollard; Jay Shendure; Martin Kircher; Nadav Ahituv

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

  • Genetically modified mouse models to help fight COVID-19
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-26
    Channabasavaiah B. Gurumurthy; Rolen M. Quadros; Guy P. Richardson; Larisa Y. Poluektova; Suzanne L. Mansour; Masato Ohtsuka

    The research community is in a race to understand the molecular mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, to repurpose currently available antiviral drugs and to develop new therapies and vaccines against coronavirus disease 2019 (COVID-19). One major challenge in achieving these goals is the paucity of suitable preclinical animal models. Mice constitute

  • Label-free capacitive assaying of biomarkers for molecular diagnostics
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-26
    Beatriz L. Garrote; Adriano Santos; Paulo R. Bueno

    The label-free analysis of biomarkers offers important advantages in developing point-of-care (PoC) biosensors. In contrast to label-based methodologies, such as ELISA, label-free analysis enables direct detection of targets without additional steps and labeled reagents. Nonetheless, label-free approaches require high sensitivity to detect the intrinsic features of a biomarker and low levels of nonspecific

  • Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-26
    Peter Møller; Amaya Azqueta; Elisa Boutet-Robinet; Gudrun Koppen; Stefano Bonassi; Mirta Milić; Goran Gajski; Solange Costa; João Paulo Teixeira; Cristiana Costa Pereira; Maria Dusinska; Roger Godschalk; Gunnar Brunborg; Kristine B. Gutzkow; Lisa Giovannelli; Marcus S. Cooke; Elke Richling; Blanca Laffon; Vanessa Valdiglesias; Nursen Basaran; Cristian Del Bo’; Bojana Zegura; Matjaz Novak; Helga Stopper;

    The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published

  • Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-23
    Juan Antonio García-León; Beatriz García-Díaz; Kristel Eggermont; Laura Cáceres-Palomo; Katrien Neyrinck; Rodrigo Madeiro da Costa; José Carlos Dávila; Anne Baron-Van Evercooren; Antonia Gutiérrez; Catherine M. Verfaillie

    Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only ~20 d, which can subsequently myelinate

  • Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-23
    Marina A. Dobrovolskaia; Kirill A. Afonin

    This protocol assesses proinflammatory properties of nucleic acid nanoparticles (NANPs) using a validated preclinical model, peripheral blood mononuclear cells (PBMCs), that is highly predictive of cytokine responses. The experimental procedure details the preparation of pyrogen-free NANPs, isolation of PBMCs from freshly collected human blood, and analysis of characteristic biomarkers (type I and

  • Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-23
    Petr Novák; Pavel Neumann; Jiří Macas

    RepeatExplorer2 is a novel version of a computational pipeline that uses graph-based clustering of next-generation sequencing reads for characterization of repetitive DNA in eukaryotes. The clustering algorithm facilitates repeat identification in any genome by using relatively small quantities of short sequence reads, and additional tools within the pipeline perform automatic annotation and quantification

  • Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-23
    Claudio Parolo; Amadeo Sena-Torralba; José Francisco Bergua; Enric Calucho; Celia Fuentes-Chust; Liming Hu; Lourdes Rivas; Ruslan Álvarez-Diduk; Emily P. Nguyen; Stefano Cinti; Daniel Quesada-González; Arben Merkoçi

    Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental

  • Tutorial: guidelines for standardized performance tests for electrodes intended for neural interfaces and bioelectronics
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-19
    Christian Boehler; Stefano Carli; Luciano Fadiga; Thomas Stieglitz; Maria Asplund

    Implantable neural interfaces advance the possibilities for neuroscientists to study the brain. They are also promising for use in a multitude of bioelectronic therapies. Electrode technology plays a central role in these developments, as the electrode surfaces form the physical interfaces between technology and the biological targets. Despite this, a common understanding of how electrodes should best

  • Intracellular neuronal recording in awake nonhuman primates
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-12
    Lixia Gao; Xiaoqin Wang

    Intracellular neuronal recordings from the brain of awake nonhuman primates have remained difficult to obtain because of several formidable technical challenges, such as poor recording stability and difficulties in maintaining long-term recording conditions. We have developed a technique to record neuronal activity by using a coaxial guide tube and sharp electrode assembly, which allows researchers

  • Jointly defining cell types from multiple single-cell datasets using LIGER
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-12
    Jialin Liu; Chao Gao; Joshua Sodicoff; Velina Kozareva; Evan Z. Macosko; Joshua D. Welch

    High-throughput single-cell sequencing technologies hold tremendous potential for defining cell types in an unbiased fashion using gene expression and epigenomic state. A key challenge in realizing this potential is integrating single-cell datasets from multiple protocols, biological contexts, and data modalities into a joint definition of cellular identity. We previously developed an approach, called

  • Light-induced synthesis of protein conjugates and its application in photoradiosynthesis of 89 Zr-radiolabeled monoclonal antibodies
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-07
    Amaury Guillou; Daniel F. Earley; Malay Patra; Jason P. Holland

    Efficient methods to functionalize proteins are essential for the development of many diagnostic and therapeutic compounds, such as fluorescent probes for immunohistochemistry, zirconium-89 radiolabeled mAbs (89Zr-mAbs) for positron emission tomography and antibody-drug conjugates (ADCs). This protocol describes a step-by-step procedure for the light-induced functionalization of proteins with compounds

  • Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks
    Nat. Protoc. (IF 10.419) Pub Date : 2020-10-01
    Michael G. Mason; José R. Botella

    The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids)

  • A generalized workflow for conducting electric field–optimized, fMRI-guided, transcranial magnetic stimulation
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-30
    Nicholas L. Balderston; Camille Roberts; Emily M. Beydler; Zhi-De Deng; Thomas Radman; Bruce Luber; Sarah H. Lisanby; Monique Ernst; Christian Grillon

    Transcranial magnetic stimulation (TMS) is a noninvasive method to stimulate the cerebral cortex that has applications in psychiatry, such as in the treatment of depression and anxiety. Although many TMS targeting methods that use figure-8 coils exist, many do not account for individual differences in anatomy or are not generalizable across target sites. This protocol combines functional magnetic resonance

  • Tutorial: a computational framework for the design and optimization of peripheral neural interfaces
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-28
    Simone Romeni; Giacomo Valle; Alberto Mazzoni; Silvestro Micera

    Peripheral neural interfaces have been successfully used in the recent past to restore sensory-motor functions in disabled subjects and for the neuromodulation of the autonomic nervous system. The optimization of these neural interfaces is crucial for ethical, clinical and economic reasons. In particular, hybrid models (HMs) constitute an effective framework to simulate direct nerve stimulation and

  • Reference-free deconvolution, visualization and interpretation of complex DNA methylation data using DecompPipeline, MeDeCom and FactorViz
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-25
    Michael Scherer; Petr V. Nazarov; Reka Toth; Shashwat Sahay; Tony Kaoma; Valentin Maurer; Nikita Vedeneev; Christoph Plass; Thomas Lengauer; Jörn Walter; Pavlo Lutsik

    DNA methylation profiling offers unique insights into human development and diseases. Often the analysis of complex tissues and cell mixtures is the only feasible option to study methylation changes across large patient cohorts. Since DNA methylomes are highly cell type specific, deconvolution methods can be used to recover cell type–specific information in the form of latent methylation components

  • Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-25
    Jianhui Nie; Qianqian Li; Jiajing Wu; Chenyan Zhao; Huan Hao; Huan Liu; Li Zhang; Lingling Nie; Haiyang Qin; Meng Wang; Qiong Lu; Xiaoyu Li; Qiyu Sun; Junkai Liu; Changfa Fan; Weijin Huang; Miao Xu; Youchun Wang

    Pseudotyped viruses are useful virological tools because of their safety and versatility. On the basis of a vesicular stomatitis virus (VSV) pseudotyped virus production system, we developed a pseudotyped virus-based neutralization assay against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in biosafety level 2 facilities. Compared with the binding antibody test, the neutralization assay

  • Solid-state 31P NMR mapping of active centers and relevant spatial correlations in solid acid catalysts.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-23
    Xianfeng Yi,Hui-Hsin Ko,Feng Deng,Shang-Bin Liu,Anmin Zheng

    Solid acid catalysts are used extensively in various advanced chemical and petrochemical processes. Their catalytic performance (namely, activity, selectivity, and reaction pathway) mostly depends on their acid properties, such as type (Brønsted versus Lewis), location, concentration, and strength, as well as the spatial correlations of their acid sites. Among the diverse methods available for acidity

  • Preparation of robust fluorescent probes for tracking endogenous formaldehyde in living cells and mouse tissue slices.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-23
    Yonghe Tang,Yuping Zhao,Weiying Lin

    Formaldehyde (FA) is the simplest active carbonyl species that can be spontaneously produced in the body and plays important roles in human cognitive ability and spatial memory. However, excessive intake of FA may cause a series of diseases, including cancer, diabetes, heart and liver diseases and various neuropathies. Hence, the exploration of sensitive and fast detection methods for FA is crucial

  • CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-21
    Baolong Xia,Gabriel Amador,Raghuvir Viswanatha,Jonathan Zirin,Stephanie E Mohr,Norbert Perrimon

    Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and

  • Establishment of patient-derived cancer organoids for drug-screening applications.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-14
    Else Driehuis,Kai Kretzschmar,Hans Clevers

    Adult stem cell–based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-derived cancer organoids therefore hold great promise for personalized medicine. Here,

  • Efficient low-cost chromatin profiling with CUT&Tag.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-10
    Hatice S Kaya-Okur,Derek H Janssens,Jorja G Henikoff,Kami Ahmad,Steven Henikoff

    We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters (‘tagmentation’) for paired-end DNA sequencing. Here, we introduce

  • Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-09
    Megan A Sheridan,Ridma C Fernando,Lucy Gardner,Michael S Hollinshead,Graham J Burton,Ashley Moffett,Margherita Y Turco

    The human placenta is essential for successful reproduction. There is great variation in the anatomy and development of the placenta in different species, meaning that animal models provide limited information about human placental development and function. Until recently, it has been impossible to isolate trophoblast cells from the human placenta that proliferate in vitro. This has limited our ability

  • Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-09
    Jianbo He,Dashuang Mo,Jingying Chen,Lingfei Luo

    RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead

  • A comprehensive guide to studying inflammasome activation and cell death.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-07
    Rebecca E Tweedell,R K Subbarao Malireddi,Thirumala-Devi Kanneganti

    Inflammasomes are multimeric heterogeneous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions

  • A standardized social preference protocol for measuring social deficits in mouse models of autism.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-07
    Benjamin Rein,Kaijie Ma,Zhen Yan

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficits and other behavioral abnormalities. The three-chamber social preference test is often used to assess social deficits in mouse models of ASD. However, varying and often contradicting phenotypic descriptions of ASD mouse models can be found in the scientific literature, and the substantial variability

  • Tutorial: structural characterization of isolated metal atoms and subnanometric metal clusters in zeolites.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-04
    Lichen Liu,Miguel Lopez-Haro,Jose J Calvino,Avelino Corma

    The encapsulation of subnanometric metal entities (isolated metal atoms and metal clusters with a few atoms) in porous materials such as zeolites can be an effective strategy for the stabilization of those metal species and therefore can be further used for a variety of catalytic reactions. However, owing to the complexity of zeolite structures and their low stability under the electron beam, it is

  • A versatile reporter system for multiplexed screening of effective epigenome editors.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-09-04
    Maria Silvia Roman Azcona,Yongxing Fang,Antonio Carusillo,Toni Cathomen,Claudio Mussolino

    The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly

  • A complete and flexible workflow for metaproteomics data analysis based on MetaProteomeAnalyzer and Prophane.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-28
    Henning Schiebenhoefer,Kay Schallert,Bernhard Y Renard,Kathrin Trappe,Emanuel Schmid,Dirk Benndorf,Katharina Riedel,Thilo Muth,Stephan Fuchs

    Metaproteomics, the study of the collective protein composition of multi-organism systems, provides deep insights into the biodiversity of microbial communities and the complex functional interplay between microbes and their hosts or environment. Thus, metaproteomics has become an indispensable tool in various fields such as microbiology and related medical applications. The computational challenges

  • A universal approach for the synthesis of mesoporous gold, palladium and platinum films for applications in electrocatalysis.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-24
    Hyunsoo Lim,Kenya Kani,Joel Henzie,Tomota Nagaura,Asep Sugih Nugraha,Muhammad Iqbal,Yong Sik Ok,Md Shahriar A Hossain,Yoshio Bando,Kevin C W Wu,Hyun-Jong Kim,Alan E Rowan,Jongbeom Na,Yusuke Yamauchi

    High-surface-area mesoporous materials expose abundant functional sites for improved performance in applications such as gas storage/separation, catalysis, and sensing. Recently, soft templates composed of amphiphilic surfactants and block copolymers have been used to introduce mesoporosity in various materials, including metals, metal oxides and carbonaceous compounds. In particular, mesoporous metals

  • Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-21
    Chuan Yan,Daniel Do,Qiqi Yang,Dalton C Brunson,John F Rawls,David M Langenau

    Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet

  • Ligand binding free-energy calculations with funnel metadynamics.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-19
    Stefano Raniolo,Vittorio Limongelli

    The accurate resolution of the binding mechanism of a ligand to its molecular target is fundamental to develop a successful drug design campaign. Free-energy calculations, which provide the energy value of the ligand–protein binding complex, are essential for resolving the binding mode of the ligand. The accuracy of free-energy calculation methods is counteracted by their poor user-friendliness, which

  • Engineering DNA nanostructures for siRNA delivery in plants.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-17
    Huan Zhang,Honglu Zhang,Gozde S Demirer,Eduardo González-Grandío,Chunhai Fan,Markita P Landry

    Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a ‘green’ alternative known as RNAi, which involves the delivery of siRNAs that downregulate

  • Imaging endocytic vesicle formation at high spatial and temporal resolutions with the pulsed-pH protocol.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-17
    Silvia Sposini,Morgane Rosendale,Léa Claverie,Thi Nhu Ngoc Van,Damien Jullié,David Perrais

    Endocytosis is a fundamental process occurring in all eukaryotic cells. Live cell imaging of endocytosis has helped to decipher many of its mechanisms and regulations. With the pulsed-pH (ppH) protocol, one can detect the formation of individual endocytic vesicles (EVs) with an unmatched temporal resolution of 2 s. The ppH protocol makes use of cargo protein (e.g., the transferrin receptor) coupled

  • Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-17
    Tetsuya Handa,Akihito Harada,Kazumitsu Maehara,Shoko Sato,Masaru Nakao,Naoki Goto,Hitoshi Kurumizaka,Yasuyuki Ohkawa,Hiroshi Kimura

    Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing

  • Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-17
    Raphael V Gayet,Helena de Puig,Max A English,Luis R Soenksen,Peter Q Nguyen,Angelo S Mao,Nicolaas M Angenent-Mari,James J Collins

    Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid–responsive materials

  • GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-14
    Erwei Zuo,Yidi Sun,Wu Wei,Tanglong Yuan,Wenqin Ying,Hao Sun,Liyun Yuan,Lars M Steinmetz,Yixue Li,Hui Yang

    Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR–Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus

  • Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-12
    Yacine Bounab,Klaus Eyer,Sophie Dixneuf,Magda Rybczynska,Cécile Chauvel,Maxime Mistretta,Trang Tran,Nathan Aymerich,Guilhem Chenon,Jean-François Llitjos,Fabienne Venet,Guillaume Monneret,Iain A Gillespie,Pierre Cortez,Virginie Moucadel,Alexandre Pachot,Alain Troesch,Philippe Leissner,Julien Textoris,Jérôme Bibette,Cyril Guyard,Jean Baudry,Andrew D Griffiths,Christophe Védrine

    Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics

  • Combined proximity labeling and affinity purification-mass spectrometry workflow for mapping and visualizing protein interaction networks.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-10
    Xiaonan Liu,Kari Salokas,Rigbe G Weldatsadik,Lisa Gawriyski,Markku Varjosalo

    Affinity purification coupled with mass spectrometry (AP–MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP−MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct

  • Direct analysis of brain phenotypes via neural blastocyst complementation.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-10
    Hai-Qiang Dai,Zhuoyi Liang,Amelia N Chang,Aimee M Chapdelaine-Williams,Beatriz Alvarado,Alex A Pollen,Frederick W Alt,Bjoern Schwer

    We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR–Cas9-mediated

  • Bacterial mock communities as standards for reproducible cytometric microbiome analysis.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-07
    Nicolas Cichocki,Thomas Hübschmann,Florian Schattenberg,Frederiek-Maarten Kerckhof,Jörg Overmann,Susann Müller

    Flow cytometry has recently established itself as a tool to track short-term dynamics in microbial community assembly and link those dynamics with ecological parameters. However, instrumental configurations of commercial cytometers and variability introduced through differential handling of the cells and instruments frequently cause data set variability at the single-cell level. This is especially

  • NAD tagSeq for transcriptome-wide identification and characterization of NAD+-capped RNAs.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-08-03
    Xiaojian Shao,Hailei Zhang,Zhu Yang,Huan Zhong,Yiji Xia,Zongwei Cai

    Several noncanonical initial nucleotides (NCINs) have been found to cap RNAs and possibly regulate RNA stability, transcription and translation. NAD+ is one of the NCINs that has recently been discovered to cap RNAs in a wide range of species. Identification of the NAD+-capped RNAs (NAD-RNAs) could help to unveil the cap-mediated regulation mechanisms. We previously reported a method termed NAD tagSeq

  • Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-31
    Katarzyna Buczak,Joanna M Kirkpatrick,Felicia Truckenmueller,Deolinda Santinha,Lino Ferreira,Stephanie Roessler,Stephan Singer,Martin Beck,Alessandro Ori

    Bottom-up mass spectrometry–based proteomics relies on protein digestion and peptide purification. The application of such methods to broadly available clinical samples such as formalin-fixed and paraffin-embedded (FFPE) tissues requires reversal of chemical crosslinking and the removal of reagents that are incompatible with mass spectrometry. Here, we describe in detail a protocol that combines tissue

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