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On-resin multicomponent protocols for biopolymer assembly and derivatization Nat. Protoc. (IF 10.419) Pub Date : 2021-01-20 Daniel G. Rivera; Manuel G. Ricardo; Aldrin V. Vasco; Ludger A. Wessjohann; Erik V. Van der Eycken
Solid-phase synthesis represents the methodological showcase for technological advances such as split-and-pool combinatorial chemistry and the automated synthesis of peptides, nucleic acids and polysaccharides. These strategies involve iterative coupling cycles that do not generate functional diversity besides that incorporated by the amino acids, nucleosides and monosaccharide building blocks. In
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Identifying genome-wide off-target sites of CRISPR RNA–guided nucleases and deaminases with Digenome-seq Nat. Protoc. (IF 10.419) Pub Date : 2021-01-18 Daesik Kim; Beum-Chang Kang; Jin-Soo Kim
Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands
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τ-SGA: synthetic genetic array analysis for systematically screening and quantifying trigenic interactions in yeast Nat. Protoc. (IF 10.419) Pub Date : 2021-01-18 Elena Kuzmin; Mahfuzur Rahman; Benjamin VanderSluis; Michael Costanzo; Chad L. Myers; Brenda J. Andrews; Charles Boone
Systematic complex genetic interaction studies have provided insight into high-order functional redundancies and genetic network wiring of the cell. Here, we describe a method for screening and quantifying trigenic interactions from ordered arrays of yeast strains grown on agar plates as individual colonies. The protocol instructs users on the trigenic synthetic genetic array analysis technique, τ-SGA
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Characterization of connective tissues using near-infrared spectroscopy and imaging Nat. Protoc. (IF 10.419) Pub Date : 2021-01-18 Isaac O. Afara; Rubina Shaikh; Ervin Nippolainen; William Querido; Jari Torniainen; Jaakko K. Sarin; Shital Kandel; Nancy Pleshko; Juha Töyräs
Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no
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Detection of aberrant gene expression events in RNA sequencing data Nat. Protoc. (IF 10.419) Pub Date : 2021-01-18 Vicente A. Yépez; Christian Mertes; Michaela F. Müller; Daniela Klaproth-Andrade; Leonhard Wachutka; Laure Frésard; Mirjana Gusic; Ines F. Scheller; Patricia F. Goldberg; Holger Prokisch; Julien Gagneur
RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects in individuals affected by genetically undiagnosed rare disorders. Pioneering studies have shown that RNA-seq could increase the diagnosis rates over DNA sequencing alone by 8–36%, depending on the disease entity and tissue probed. To accelerate adoption of RNA-seq by human genetics centers
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Precision genome editing using cytosine and adenine base editors in mammalian cells Nat. Protoc. (IF 10.419) Pub Date : 2021-01-18 Tony P. Huang; Gregory A. Newby; David R. Liu
Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively
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Multimodal detection of protein isoforms and nucleic acids from mouse pre-implantation embryos Nat. Protoc. (IF 10.419) Pub Date : 2021-01-15 Elisabet Rosàs-Canyelles; Andrew J. Modzelewski; Alisha Geldert; Lin He; Amy E. Herr
Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo
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Mapping mechanical properties of biological materials via an add-on Brillouin module to confocal microscopes Nat. Protoc. (IF 10.419) Pub Date : 2021-01-15 Jitao Zhang; Giuliano Scarcelli
Several techniques have been developed over the past few decades to assess the mechanical properties of biological samples, which has fueled a rapid growth in the fields of biophysics, bioengineering, and mechanobiology. In this context, Brillouin optical spectroscopy has long been known as an intriguing modality for noncontact material characterization. However, limited by speed and sample damage
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Facile synthesis of per(6- O - tert -butyldimethylsilyl)-α-, β-, and γ-cyclodextrin as protected intermediates for the functionalization of the secondary face of the macrocycles Nat. Protoc. (IF 10.419) Pub Date : 2021-01-15 Gábor Benkovics; Milo Malanga; Giovanna Cutrone; Szabolcs Béni; Antonio Vargas-Berenguel; Juan Manuel Casas-Solvas
Per(6-O-tert-butyldimethylsilyl)-α-, β- and γ-cyclodextrin derivatives are well-known as synthetic intermediates that enable the selective mono-, partial, or perfunctionalization of the secondary face of the macrocycles. Although silylation of the primary rim is readily achieved by treatment with tert-butyldimethylsilyl chloride in the presence of pyridine (either alone or mixed with a co-solvent)
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Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables Nat. Protoc. (IF 10.419) Pub Date : 2021-01-15 Edward Geeurickx; Lien Lippens; Pekka Rappu; Bruno G. De Geest; Olivier De Wever; An Hendrix
The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have
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Author Correction: A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents Nat. Protoc. (IF 10.419) Pub Date : 2021-01-14 Mattia Privitera; Kim David Ferrari; Lukas M. von Ziegler; Oliver Sturman; Sian N. Duss; Amalia Floriou-Servou; Pierre-Luc Germain; Yannick Vermeiren; Matthias T. Wyss; Peter P. De Deyn; Bruno Weber; Johannes Bohacek
A Correction to this paper has been published: https://doi.org/10.1038/s41596-021-00493-6
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Author Correction: Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap Nat. Protoc. (IF 10.419) Pub Date : 2021-01-13 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
A Correction to this paper has been published: https://doi.org/10.1038/s41596-021-00492-7.
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High-throughput cultivation and identification of bacteria from the plant root microbiota Nat. Protoc. (IF 10.419) Pub Date : 2021-01-13 Jingying Zhang; Yong-Xin Liu; Xiaoxuan Guo; Yuan Qin; Ruben Garrido-Oter; Paul Schulze-Lefert; Yang Bai
Cultivating native bacteria from roots of plants grown in a given environment is essential for dissecting the functions of the root microbiota for plant growth and health with strain-specific resolution. In this study, we established a straightforward protocol for high-throughput bacterial isolation from fresh root samples using limiting dilution to ensure that most cultured bacteria originated from
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Decoding personal biotic and abiotic airborne exposome Nat. Protoc. (IF 10.419) Pub Date : 2021-01-13 Chao Jiang; Xinyue Zhang; Peng Gao; Qiong Chen; Michael Snyder
The complexity and dynamics of human diseases are driven by the interactions between internal molecular activities and external environmental exposures. Although advances in omics technology have dramatically broadened the understanding of internal molecular and cellular mechanisms, understanding of the external environmental exposures, especially at the personal level, is still rudimentary in comparison
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Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells Nat. Protoc. (IF 10.419) Pub Date : 2021-01-13 Sarah Kit Leng Lui; Sarah Keegan; Peter Tonzi; Malik Kahli; Yu-Hung Chen; Noor Chalhoub; Kate E. Coleman; David Fenyo; Duncan J. Smith; Tony T. Huang
The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the
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Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy Nat. Protoc. (IF 10.419) Pub Date : 2021-01-13 Siddarth Narasimhan; Cecilia Pinto; Alessandra Lucini Paioni; Johan van der Zwan; Gert E. Folkers; Marc Baldus
For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular
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Author Correction: Establishment of patient-derived cancer organoids for drug-screening applications Nat. Protoc. (IF 10.419) Pub Date : 2021-01-12 Else Driehuis; Kai Kretzschmar; Hans Clevers
A Correction to this paper has been published: https://doi.org/10.1038/s41596-021-00494-5
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A robust unsupervised machine-learning method to quantify the morphological heterogeneity of cells and nuclei Nat. Protoc. (IF 10.419) Pub Date : 2021-01-11 Jude M. Phillip; Kyu-Sang Han; Wei-Chiang Chen; Denis Wirtz; Pei-Hsun Wu
Cell morphology encodes essential information on many underlying biological processes. It is commonly used by clinicians and researchers in the study, diagnosis, prognosis, and treatment of human diseases. Quantification of cell morphology has seen tremendous advances in recent years. However, effectively defining morphological shapes and evaluating the extent of morphological heterogeneity within
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Stem-cell-derived human microglia transplanted into mouse brain to study human disease Nat. Protoc. (IF 10.419) Pub Date : 2021-01-11 Nicola Fattorelli; Anna Martinez-Muriana; Leen Wolfs; Ivana Geric; Bart De Strooper; Renzo Mancuso
Microglia are critically involved in complex neurological disorders with a strong genetic component, such as Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia. Although mouse microglia can recapitulate aspects of human microglia physiology, they do not fully capture the human genetic aspects of disease and do not reproduce all human cell states. Primary cultures of human microglia
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Reaction screening in multiwell plates: high-throughput optimization of a Buchwald–Hartwig amination Nat. Protoc. (IF 10.419) Pub Date : 2021-01-11 Adam Cook; Roxanne Clément; Stephen G. Newman
Chemical space is vast, and chemical reactions involve the complex interplay of multiple variables. As a consequence, reactions can fail for subtle reasons, necessitating screening of conditions. High-throughput experimentation (HTE) techniques enable a more comprehensive array of data to be obtained in a relatively short amount of time. Although HTE can be most efficiently achieved with automated
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Analysis framework and experimental design for evaluating synergy-driving gene expression Nat. Protoc. (IF 10.419) Pub Date : 2021-01-11 Nadine Schrode; Carina Seah; P. J. Michael Deans; Gabriel Hoffman; Kristen J. Brennand
The mechanisms by which genetic risk variants interact with each other, as well as environmental factors, to contribute to complex genetic disorders remain unclear. We describe in detail our recently published approach to resolve distinct additive and synergistic transcriptomic effects after combinatorial manipulation of genetic variants and/or chemical perturbagens. Although first developed for CRISPR-based
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Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells Nat. Protoc. (IF 10.419) Pub Date : 2021-01-11 Hiroyuki Koike; Kentaro Iwasawa; Rie Ouchi; Mari Maezawa; Masaki Kimura; Asuka Kodaka; Shozo Nishii; Wendy L. Thompson; Takanori Takebe
Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring
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OCTAD: an open workspace for virtually screening therapeutics targeting precise cancer patient groups using gene expression features Nat. Protoc. (IF 10.419) Pub Date : 2020-12-23 Billy Zeng; Benjamin S. Glicksberg; Patrick Newbury; Evgeny Chekalin; Jing Xing; Ke Liu; Anita Wen; Caven Chow; Bin Chen
As the field of precision medicine progresses, treatments for patients with cancer are starting to be tailored to their molecular as well as their clinical features. The emerging cancer subtypes defined by these molecular features require that dedicated resources be used to assist the discovery of drug candidates for preclinical evaluation. Voluminous gene expression profiles of patients with cancer
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An unbiased method for evaluating the genome-wide specificity of base editors in rice Nat. Protoc. (IF 10.419) Pub Date : 2020-12-21 Shuai Jin; Qiang Gao; Caixia Gao
Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe
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Labeling and tracking of immune cells in ex vivo human skin Nat. Protoc. (IF 10.419) Pub Date : 2020-12-21 Feline E. Dijkgraaf; Mireille Toebes; Mark Hoogenboezem; Marjolijn Mertz; David W. Vredevoogd; Tiago R. Matos; Marcel B. M. Teunissen; Rosalie M. Luiten; Ton N. Schumacher
Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other
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High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS Nat. Protoc. (IF 10.419) Pub Date : 2020-12-21 Anna Biernacka; Magdalena Skrzypczak; Yingjie Zhu; Philippe Pasero; Maga Rowicka; Krzysztof Ginalski
DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such
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Design, assembly, and characterization of membrane-spanning DNA nanopores Nat. Protoc. (IF 10.419) Pub Date : 2020-12-21 Conor Lanphere; Daniel Offenbartl-Stiegert; Adam Dorey; Genevieve Pugh; Elena Georgiou; Yongzheng Xing; Jonathan R. Burns; Stefan Howorka
DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as
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A surgical mouse model of neonatal pressure overload by transverse aortic constriction Nat. Protoc. (IF 10.419) Pub Date : 2020-12-16 Mona Malek Mohammadi; Aya Abouissa; Joerg Heineke
Cardiac disease is the main cause of death worldwide. Insufficient regeneration of the adult mammalian heart is a major driver of cardiac morbidity and mortality. Cardiac regeneration occurs in early postnatal mice, thus understanding mechanisms of mammalian cardiac regeneration could facilitate the development of novel therapeutic strategies. Here, we provide a detailed description of a neonatal mouse
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Implementation of a 4Pi-SMS super-resolution microscope Nat. Protoc. (IF 10.419) Pub Date : 2020-12-16 Jingyu Wang; Edward S. Allgeyer; George Sirinakis; Yongdeng Zhang; Kevin Hu; Mark D. Lessard; Yiming Li; Robin Diekmann; Michael A. Phillips; Ian M. Dobbie; Jonas Ries; Martin J. Booth; Joerg Bewersdorf
The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated
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Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development Nat. Protoc. (IF 10.419) Pub Date : 2020-12-16 Stefano L. Giandomenico; Magdalena Sutcliffe; Madeline A. Lancaster
Cerebral organoids, or brain organoids, can be generated from a wide array of emerging technologies for modeling brain development and disease. The fact that they are cultured in vitro makes them easily accessible both genetically and for live assays such as fluorescence imaging. In this Protocol Extension, we describe a modified version of our original protocol (published in 2014) that can be used
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Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing Nat. Protoc. (IF 10.419) Pub Date : 2020-12-14 Peter Ellis; Luiza Moore; Mathijs A. Sanders; Timothy M. Butler; Simon F. Brunner; Henry Lee-Six; Robert Osborne; Ben Farr; Tim H. H. Coorens; Andrew R. J. Lawson; Alex Cagan; Mike R. Stratton; Inigo Martincorena; Peter J. Campbell
Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input
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Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes Nat. Protoc. (IF 10.419) Pub Date : 2020-12-14 Ruoxiao Xie; Zhe Liang; Yongjian Ai; Wenchen Zheng; Jialiang Xiong; Peidi Xu; Yupeng Liu; Mingyu Ding; Jianyi Gao; Jiaping Wang; Qionglin Liang
Microtissues with specific structures and integrated vessels play a key role in maintaining organ functions. To recapitulate the in vivo environment for tissue engineering and organ-on-a-chip purposes, it is essential to develop perfusable biomimetic microscaffolds. We developed facile all-aqueous microfluidic approaches for producing perfusable hydrogel microtubes with diverse biomimetic sizes and
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Inducible mouse models of colon cancer for the analysis of sporadic and inflammation-driven tumor progression and lymph node metastasis Nat. Protoc. (IF 10.419) Pub Date : 2020-12-14 Clemens Neufert; Christina Heichler; Thomas Brabletz; Kristina Scheibe; Verawan Boonsanay; Florian R. Greten; Markus F. Neurath
Despite advances in the detection and therapy of colorectal cancer (CRC) in recent years, CRC has remained a major challenge in clinical practice. Although alternative methods for modeling CRC have been developed, animal models of CRC remain helpful when analyzing molecular aspects of pathogenesis and are often used to perform preclinical in vivo studies of potential therapeutics. This protocol updates
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High-speed super-resolution imaging of rotationally symmetric structures using SPEED microscopy and 2D-to-3D transformation Nat. Protoc. (IF 10.419) Pub Date : 2020-12-14 Yichen Li; Mark Tingey; Andrew Ruba; Weidong Yang
Various super-resolution imaging techniques have been developed to break the diffraction-limited resolution of light microscopy. However, it still remains challenging to obtain three-dimensional (3D) super-resolution information of structures and dynamic processes in live cells at high speed. We recently developed high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy and its two-dimensional
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Responsive and activable nanomedicines for remodeling the tumor microenvironment Nat. Protoc. (IF 10.419) Pub Date : 2020-12-11 Yinlong Zhang; Xuexiang Han; Guangjun Nie
Here we describe two protocols for the construction of responsive and activable nanomedicines that regulate the tumor microenvironment (TME). The TME is composed of all non-cellular and cellular components surrounding a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix and has a crucial role in tumor initiation, growth, and metastasis
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Generation of neighbor-labeling cells to study intercellular interactions in vivo Nat. Protoc. (IF 10.419) Pub Date : 2020-12-11 Luigi Ombrato; Emma Nolan; Diana Passaro; Ivana Kurelac; Victoria L. Bridgeman; Alexander Waclawiczek; Delfim Duarte; Cristina Lo Celso; Dominique Bonnet; Ilaria Malanchi
Understanding cell–cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding
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A microfluidics-based stem cell model of early post-implantation human development Nat. Protoc. (IF 10.419) Pub Date : 2020-12-11 Yi Zheng; Yue Shao; Jianping Fu
Early post-implantation human embryonic development has been challenging to study due to both technical limitations and ethical restrictions. Proper modeling of the process is important for infertility and toxicology research. Here we provide details of the design and implementation of a microfluidic device that can be used to model human embryo development. The microfluidic human embryo model is established
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Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions Nat. Protoc. (IF 10.419) Pub Date : 2020-12-11 Kang Soo Lee; Fátima C. Pereira; Márton Palatinszky; Lars Behrendt; Uria Alcolombri; David Berry; Michael Wagner; Roman Stocker
Stable isotope labeling of microbial taxa of interest and their sorting provide an efficient and direct way to answer the question “who does what?” in complex microbial communities when coupled with fluorescence in situ hybridization or downstream ‘omics’ analyses. We have developed a platform for automated Raman-based sorting in which optical tweezers and microfluidics are used to sort individual
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Imaging and optogenetic modulation of vascular mural cells in the live brain Nat. Protoc. (IF 10.419) Pub Date : 2020-12-09 Lei Tong; Robert A. Hill; Eyiyemisi C. Damisah; Katie N. Murray; Peng Yuan; Angelique Bordey; Jaime Grutzendler
Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood–brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for
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In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels Nat. Protoc. (IF 10.419) Pub Date : 2020-12-09 Samuel S. Hinman; Yuli Wang; Raehyun Kim; Nancy L. Allbritton
The large intestine, with its array of crypts lining the epithelium and diverse luminal contents, regulates homeostasis throughout the body. In vitro crypts formed from primary human intestinal epithelial stem cells on a 3D shaped hydrogel scaffold replicate the functional and architectural features of in vivo crypts. Collagen scaffolding assembly methods are provided, along with the microfabrication
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Digital postprocessing and image segmentation for objective analysis of colorimetric reactions Nat. Protoc. (IF 10.419) Pub Date : 2020-12-09 M. Shane Woolf; Leah M. Dignan; Anchi T. Scott; James P. Landers
Recently, there has been an explosion of scientific literature describing the use of colorimetry for monitoring the progression or the endpoint result of colorimetric reactions. The availability of inexpensive imaging technology (e.g., scanners, Raspberry Pi, smartphones and other sub-$50 digital cameras) has lowered the barrier to accessing cost-efficient, objective detection methodologies. However
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Tracking endocytosis and intracellular distribution of spherical nucleic acids with correlative single-cell imaging Nat. Protoc. (IF 10.419) Pub Date : 2020-12-07 Mengmeng Liu; Fei Wang; Xueli Zhang; Xiuhai Mao; Lihua Wang; Yang Tian; Chunhai Fan; Qian Li
A comprehensive understanding of interactions between nanoparticles (NPs) and biological components is critical to the clinical application of NPs and nanomedicine. Here we provide a step-by-step correlative imaging approach to investigate plasmonic NPs of different aggregation states at the single-cell level. Traceable spherical nucleic acids (SNAs) are fabricated by decorating 50-nm spherical gold
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Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data Nat. Protoc. (IF 10.419) Pub Date : 2020-12-07 Tallulah S. Andrews; Vladimir Yu Kiselev; Davis McCarthy; Martin Hemberg
Single-cell RNA sequencing (scRNA-seq) is a popular and powerful technology that allows you to profile the whole transcriptome of a large number of individual cells. However, the analysis of the large volumes of data generated from these experiments requires specialized statistical and computational methods. Here we present an overview of the computational workflow involved in processing scRNA-seq
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Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence Nat. Protoc. (IF 10.419) Pub Date : 2020-12-07 Marc Luginbühl; Frederike Stöth; Alexandra Schröck; Stefan Gaugler; Wolfgang Weinmann
Phosphatidylethanol (PEth), which is formed by enzymatic reaction between ethanol and phosphatidylcholine, is a direct marker for alcohol usage. PEth has a long elimination half-life (~5–10 d) and specimens can be sampled using minimally invasive microsampling strategies. In combination with rapid analysis procedures PEth has proved to be advantageous for the detection of abstinence over other direct
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Tools for experimental and computational analyses of off-target editing by programmable nucleases Nat. Protoc. (IF 10.419) Pub Date : 2020-12-07 X. Robert Bao; Yidan Pan; Ciaran M. Lee; Timothy H. Davis; Gang Bao
Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental
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Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells Nat. Protoc. (IF 10.419) Pub Date : 2020-12-04 Jiaxuan Chen; Aleksandra Ellert-Miklaszewska; Stefano Garofalo; Arindam K. Dey; Jingjie Tang; Yifan Jiang; Flora Clément; Patrice N. Marche; Xiaoxuan Liu; Bozena Kaminska; Angela Santoni; Cristina Limatola; John J. Rossi; Jiehua Zhou; Ling Peng
Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune
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Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome Nat. Protoc. (IF 10.419) Pub Date : 2020-12-04 Dylan G. Maghini; Eli L. Moss; Summer E. Vance; Ami S. Bhatt
Short-read metagenomic sequencing and de novo genome assembly of the human gut microbiome can yield draft bacterial genomes without isolation and culture. However, bacterial genomes assembled from short-read sequencing are often fragmented. Furthermore, these metagenome-assembled genomes often exclude repeated genomic elements, such as mobile genetic elements, compromising our understanding of the
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Inducing different severities of traumatic brain injury in Drosophila using a piezoelectric actuator Nat. Protoc. (IF 10.419) Pub Date : 2020-12-04 Janani Saikumar; Joshua Kim; China N. Byrns; Matthew Hemphill; David F. Meaney; Nancy M. Bonini
Drosophila models have been instrumental in providing insights into molecular mechanisms of neurodegeneration, with wide application to human disease. The brain degeneration associated with traumatic brain injury (TBI) has been modeled in Drosophila using devices that inflict trauma on multiple parts of the fly body, including the head. However, the injuries produced by these models are not specific
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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