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  • 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

  • Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-31
    Erin E Diel,Jeff W Lichtman,Douglas S Richardson

    Spherical aberration (SA) occurs when light rays entering at different points of a spherical lens are not focused to the same point of the optical axis. SA that occurs inside the lens elements of a fluorescence microscope is well understood and corrected for. However, SA is also induced when light passes through an interface of refractive index (RI)-mismatched substances (i.e., a discrepancy between

  • Standardized bacteriophage purification for personalized phage therapy.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-24
    Tiffany Luong,Ann-Charlott Salabarria,Robert A Edwards,Dwayne R Roach

    The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward—by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational

  • Tutorial: a guide to performing polygenic risk score analyses.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-24
    Shing Wan Choi,Timothy Shin-Heng Mak,Paul F O'Reilly

    A polygenic score (PGS) or polygenic risk score (PRS) is an estimate of an individual’s genetic liability to a trait or disease, calculated according to their genotype profile and relevant genome-wide association study (GWAS) data. While present PRSs typically explain only a small fraction of trait variance, their correlation with the single largest contributor to phenotypic variation—genetic liability—has

  • A graphical user interface to design high-throughput optogenetic experiments with the optoPlate-96.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-24
    Oliver S Thomas,Maximilian Hörner,Wilfried Weber

  • Organotypic culture assays for murine and human primary and metastatic-site tumors.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-20
    Veena Padmanaban,Eloise M Grasset,Neil M Neumann,Andrew K Fraser,Elodie Henriet,William Matsui,Phuoc T Tran,Kevin J Cheung,Dan Georgess,Andrew J Ewald

    Cancer invasion and metastasis are challenging to study in vivo since they occur deep inside the body over extended time periods. Organotypic 3D culture of fresh tumor tissue enables convenient real-time imaging, genetic and microenvironmental manipulation and molecular analysis. Here, we provide detailed protocols to isolate and culture heterogenous organoids from murine and human primary and metastatic

  • A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-20
    Ling Fu,Zongmin Li,Keke Liu,Caiping Tian,Jixiang He,Jingyang He,Fuchu He,Ping Xu,Jing Yang

    Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms

  • Complex-centric proteome profiling by SEC-SWATH-MS for the parallel detection of hundreds of protein complexes.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-20
    Isabell Bludau,Moritz Heusel,Max Frank,George Rosenberger,Robin Hafen,Amir Banaei-Esfahani,Audrey van Drogen,Ben C Collins,Matthias Gstaiger,Ruedi Aebersold

    Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules are therefore of major significance in basic and translational biology. However, detection and quantification of

  • Genome-wide piggyBac transposon-based mutagenesis and quantitative insertion-site analysis in haploid Candida species.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-17
    Zeyao Li,Haitao Wang,Chunling Cai,Ada Hang-Heng Wong,Jianbin Wang,Jiaxin Gao,Yue Wang

    Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which

  • In vivo compression and imaging in mouse brain to measure the effects of solid stress.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-17
    Hadi T Nia,Meenal Datta,Giorgio Seano,Sue Zhang,William W Ho,Sylvie Roberge,Peigen Huang,Lance L Munn,Rakesh K Jain

    We recently developed an in vivo compression device that simulates the solid mechanical forces exerted by a growing tumor on the surrounding brain tissue and delineates the physical versus biological effects of a tumor. This device, to our knowledge the first of its kind, can recapitulate the compressive forces on the cerebellar cortex from primary (e.g., glioblastoma) and metastatic (e.g., breast

  • Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-17
    Qiongyu Li,Yixuan Xie,Maurice Wong,Mariana Barboza,Carlito B Lebrilla

    The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in

  • Large-scale site-specific mapping of the O-GalNAc glycoproteome.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-17
    Weiming Yang,Angellina Song,Minghui Ao,Yuanwei Xu,Hui Zhang

    Protein glycosylation is one of the most common protein modifications. A major type of protein glycosylation is O-GalNAcylation, in which GalNAc-type glycans are attached to protein Ser or Thr residues via an O-linked glycosidic bond. O-GalNAcylation is thought to play roles in protein folding, stability, trafficking and protein interactions, and identification of the site-specific O-GalNAc glycoproteome

  • Identifying unknown metabolites using NMR-based metabolic profiling techniques.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-17
    Isabel Garcia-Perez,Joram M Posma,Jose Ivan Serrano-Contreras,Claire L Boulangé,Queenie Chan,Gary Frost,Jeremiah Stamler,Paul Elliott,John C Lindon,Elaine Holmes,Jeremy K Nicholson

    Metabolic profiling of biological samples provides important insights into multiple physiological and pathological processes but is hindered by a lack of automated annotation and standardized methods for structure elucidation of candidate disease biomarkers. Here we describe a system for identifying molecular species derived from nuclear magnetic resonance (NMR) spectroscopy-based metabolic phenotyping

  • A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-17
    Takashi Umehara,Natsumi Tsujita,Zhendong Zhu,Moeka Ikedo,Masayuki Shimada

    The preferred sex of livestock differs among breeders; for example, dairy farmers prefer female calves for the production of milk, whereas cattle meat producers often prefer males. Sexing of laboratory animals is also beneficial in some research fields, including reproductive biology and metabolic studies. Most sexing methods separate X sperm and Y sperm with a cell sorter. Here, we describe a system

  • Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-15
    Tao Zhang,Taoran Tian,Ronghui Zhou,Songhang Li,Wenjuan Ma,Yuxin Zhang,Nanxin Liu,Sirong Shi,Qianshun Li,Xueping Xie,Yichen Ge,Mengting Liu,Qi Zhang,Shiyu Lin,Xiaoxiao Cai,Yunfeng Lin

    Although organic nanomaterials and inorganic nanoparticles possess inherent flexibility, facilitating functional modification, increased intracellular uptake and controllable drug release, their underlying cytotoxicity and lack of specificity still cause safety concerns. Owing to their merits, which include natural biocompatibility, structural stability, unsurpassed programmability, ease of internalization

  • Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS).
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-10
    Eneko Villanueva,Tom Smith,Rayner M L Queiroz,Mie Monti,Mariavittoria Pizzinga,Mohamed Elzek,Veronica Dezi,Robert F Harvey,Manasa Ramakrishna,Anne E Willis,Kathryn S Lilley

    RNA−protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA−protein adducts

  • CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-10
    Yaojun Tong,Christopher M Whitford,Kai Blin,Tue S Jørgensen,Tilmann Weber,Sang Yup Lee

    Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive

  • lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-08
    M Grace Gordon,Fumitaka Inoue,Beth Martin,Max Schubach,Vikram Agarwal,Sean Whalen,Shiyun Feng,Jingjing Zhao,Tal Ashuach,Ryan Ziffra,Anat Kreimer,Ilias Georgakopoulous-Soares,Nir Yosef,Chun Jimmie Ye,Katherine S Pollard,Jay Shendure,Martin Kircher,Nadav Ahituv

    Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence

  • A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-06
    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

    The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established

  • Geometric morphometrics of microscopic animals as exemplified by model nematodes.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-06
    Tobias Theska,Bogdan Sieriebriennikov,Sara S Wighard,Michael S Werner,Ralf J Sommer

    While a host of molecular techniques are utilized by evolutionary developmental (evo-devo) biologists, tools for quantitative evaluation of morphology are still largely underappreciated, especially in studies on microscopic animals. Here, we provide a standardized protocol for geometric morphometric analyses of 2D landmark data sets using a combination of the geomorph and Morpho R packages. Furthermore

  • Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-01
    Marie-Theres Gansauge,Ayinuer Aximu-Petri,Sarah Nagel,Matthias Meyer

    It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3′ ends of single DNA strands, the synthesis of a complementary strand

  • Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-26
    Pavel V Bashkirov,Peter I Kuzmin,Ksenia Chekashkina,Pedro Arrasate,Javier Vera Lillo,Anna V Shnyrova,Vadim A Frolov

    Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which

  • FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-26
    Alba Font-Tello,Nikolas Kesten,Yingtian Xie,Len Taing,Damir Varešlija,Leonie S Young,Anis A Hamid,Eliezer M Van Allen,Christopher J Sweeney,Evisa Gjini,Ana Lako,F Steven Hodi,Joaquim Bellmunt,Myles Brown,Paloma Cejas,Henry W Long

    Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized

  • Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography-mass spectrometry.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-24
    Fujian Zheng,Xinjie Zhao,Zhongda Zeng,Lichao Wang,Wangjie Lv,Qingqing Wang,Guowang Xu

    Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability

  • Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-22
    Dekai Ye,Min Li,Tingting Zhai,Ping Song,Lu Song,Hua Wang,Xiuhai Mao,Fei Wang,Xueli Zhang,Zhilei Ge,Jiye Shi,Lihua Wang,Chunhai Fan,Qian Li,Xiaolei Zuo

    Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze

  • A scalable SCENIC workflow for single-cell gene regulatory network analysis.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-19
    Bram Van de Sande,Christopher Flerin,Kristofer Davie,Maxime De Waegeneer,Gert Hulselmans,Sara Aibar,Ruth Seurinck,Wouter Saelens,Robrecht Cannoodt,Quentin Rouchon,Toni Verbeiren,Dries De Maeyer,Joke Reumers,Yvan Saeys,Stein Aerts

    This protocol explains how to perform a fast SCENIC analysis alongside standard best practices steps on single-cell RNA-sequencing data using software containers and Nextflow pipelines. SCENIC reconstructs regulons (i.e., transcription factors and their target genes) assesses the activity of these discovered regulons in individual cells and uses these cellular activity patterns to find meaningful clusters

  • Macrophage phagocytosis assay with reconstituted target particles.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-19
    Aaron M Joffe,Matthew H Bakalar,Daniel A Fletcher

    Macrophage phagocytosis can be triggered by diverse receptor-ligand interactions to clear pathogens and dead cells from a host. Many ways of assaying phagocytosis exist that utilize a variety of phagocytic targets with different combinations of receptor-ligand interactions, making comparisons difficult. To study how phagocytosis is affected by specific changes to the target surface, we developed an

  • COVIDep: a web-based platform for real-time reporting of vaccine target recommendations for SARS-CoV-2.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-07-01
    Syed Faraz Ahmed,Ahmed A Quadeer,Matthew R McKay

  • Tutorial: multivariate classification for vibrational spectroscopy in biological samples.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-17
    Camilo L M Morais,Kássio M G Lima,Maneesh Singh,Francis L Martin

    Vibrational spectroscopy techniques, such as Fourier-transform infrared (FTIR) and Raman spectroscopy, have been successful methods for studying the interaction of light with biological materials and facilitating novel cell biology analysis. Spectrochemical analysis is very attractive in disease screening and diagnosis, microbiological studies and forensic and environmental investigations because of

  • High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-15
    Elena G Shcherbakova,Tony D James,Pavel Anzenbacher

    Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines

  • Analysis of task-based functional MRI data preprocessed with fMRIPrep.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-08
    Oscar Esteban,Rastko Ciric,Karolina Finc,Ross W Blair,Christopher J Markiewicz,Craig A Moodie,James D Kent,Mathias Goncalves,Elizabeth DuPre,Daniel E P Gomez,Zhifang Ye,Taylor Salo,Romain Valabregue,Inge K Amlien,Franziskus Liem,Nir Jacoby,Hrvoje Stojić,Matthew Cieslak,Sebastian Urchs,Yaroslav O Halchenko,Satrajit S Ghosh,Alejandro De La Vega,Tal Yarkoni,Jessey Wright,William H Thompson,Russell A Poldrack

    Functional magnetic resonance imaging (fMRI) is a standard tool to investigate the neural correlates of cognition. fMRI noninvasively measures brain activity, allowing identification of patterns evoked by tasks performed during scanning. Despite the long history of this technique, the idiosyncrasies of each dataset have led to the use of ad-hoc preprocessing protocols customized for nearly every different

  • Ensuring accurate resource identification.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-06-03

    Nature Protocols is pleased to be a part of the Resource Identification Initiative, a project aimed at improving the reproducibility of research by clearly identifying key biological resources. Stable unique digital identifiers, called Research Resource Identifiers (RRIDs), are assigned to individual resources, allowing users to accurately identify and source them, track their history, identify known

  • Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques.
    Nat. Protoc. (IF 10.419) Pub Date : 2020-05-25
    Tina Uroda,Isabel Chillón,Paolo Annibale,Jean-Marie Teulon,Ombeline Pessey,Manikandan Karuppasamy,Jean-Luc Pellequer,Marco Marcia

    Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution

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