2,681 results match your criteria Nature protocols[Journal]


Fractionation of lignocellulosic biomass to produce uncondensed aldehyde-stabilized lignin.

Nat Protoc 2019 Feb 18. Epub 2019 Feb 18.

Laboratory of Sustainable and Catalytic Processing, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Lignin is one of the most promising sources of renewable aromatic hydrocarbons. Current methods for its extraction from lignocellulosic biomass-which include the kraft, sulfite, and organosolv processes-result in the rapid formation of carbon-carbon bonds, leading to a condensed lignin that cannot be effectively depolymerized into its constituent monomers. Treatment of lignocellulosic biomass with aldehydes during lignin extraction generates an aldehyde-stabilized lignin that is uncondensed and can be converted into its monomers at near-theoretical yields. Read More

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http://dx.doi.org/10.1038/s41596-018-0121-7DOI Listing
February 2019

A practical guide to optimization in X10 expansion microscopy.

Nat Protoc 2019 Feb 18. Epub 2019 Feb 18.

Institute of Science and Technology Austria, Klosterneuburg, Austria.

Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60-80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Read More

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http://www.nature.com/articles/s41596-018-0117-3
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http://dx.doi.org/10.1038/s41596-018-0117-3DOI Listing
February 2019
1 Read

Publisher Correction: Live imaging of stem cells in the germarium of the Drosophila ovary using a reusable gas-permeable imaging chamber.

Nat Protoc 2019 Feb 15. Epub 2019 Feb 15.

Department of Biomedical Engineering, Columbia University, New York, NY, USA.

The version of this paper originally published contained an incorrect unit abbreviation in Step 21: "0.20 g/mL" should have been "0.20 mg/mL. Read More

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http://www.nature.com/articles/s41596-019-0134-x
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http://dx.doi.org/10.1038/s41596-019-0134-xDOI Listing
February 2019
2 Reads

Using the tube test to measure social hierarchy in mice.

Nat Protoc 2019 Feb 15. Epub 2019 Feb 15.

Interdisciplinary Institute of Neuroscience and Technology, Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China.

Investigation of the neural mechanisms underlying social hierarchy requires a reliable and effective behavioral test. The tube test is a simple and robust behavioral assay that we recently validated as a reliable measure of social hierarchy in mice. The test was demonstrated to produce results largely consistent with the results seen when using other dominance measures, including the warm spot test, territory urine marking or the courtship ultrasound vocalization test. Read More

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http://dx.doi.org/10.1038/s41596-018-0116-4DOI Listing
February 2019

Using CAPTURE to detect spacer acquisition in native CRISPR arrays.

Nat Protoc 2019 Feb 11. Epub 2019 Feb 11.

Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.

CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 10 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. Read More

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http://dx.doi.org/10.1038/s41596-018-0123-5DOI Listing
February 2019

Design, execution, and analysis of CRISPR-Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans.

Nat Protoc 2019 Feb 8. Epub 2019 Feb 8.

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.

The study of fungal pathogens is of immediate importance, yet progress is hindered by the technical challenges of genetic manipulation. For Candida species, their inability to maintain plasmids, unusual codon usage, and inefficient homologous recombination are among the obstacles limiting efficient genetic manipulation. New advances in genomic biotechnologies-particularly CRISPR-based tools-have revolutionized genome editing for many fungal species. Read More

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http://dx.doi.org/10.1038/s41596-018-0122-6DOI Listing
February 2019

Multiplexed profiling of RNA and protein expression signatures in individual cells using flow or mass cytometry.

Nat Protoc 2019 Feb 6. Epub 2019 Feb 6.

Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.

Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. RNA within single cells can be analyzed using unbiased sequencing protocols or through more targeted approaches using in situ hybridization (ISH). The proximity ligation assay for RNA (PLAYR) approach is a sensitive and high-throughput technique that relies on in situ and proximal ligation to measure at least 27 specific RNAs by flow or mass cytometry. Read More

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http://dx.doi.org/10.1038/s41596-018-0120-8DOI Listing
February 2019
1 Read

Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute.

Nat Protoc 2019 Feb 1. Epub 2019 Feb 1.

Shanghai Key Laboratory of Tuberculosis, Clinical Translational Research Center, Shanghai Pulmonary Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. Read More

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http://www.nature.com/articles/s41596-018-0113-7
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http://dx.doi.org/10.1038/s41596-018-0113-7DOI Listing
February 2019
20 Reads

Pipelines for cross-species and genome-wide prediction of long noncoding RNA binding.

Nat Protoc 2019 Jan 30. Epub 2019 Jan 30.

Bioinformatics Section, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

Abundant long, noncoding RNAs (lncRNAs) in mammals can bind to DNA sequences and recruit histone- and DNA-modifying enzymes to binding sites to epigenetically regulate target genes. However, most lncRNAs' binding motifs and target sites are unknown. The large numbers of lncRNAs and target sites in the whole genome make it infeasible to examine lncRNA binding to DNA purely experimentally. Read More

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http://dx.doi.org/10.1038/s41596-018-0115-5DOI Listing
January 2019

Micropipette force sensors for in vivo force measurements on single cells and multicellular microorganisms.

Nat Protoc 2019 Feb;14(2):594-615

Max Planck Institute for Dynamics and Self-Organization (MPIDS), Göttingen, Germany.

Measuring forces from the piconewton to millinewton range is of great importance for the study of living systems from a biophysical perspective. The use of flexible micropipettes as highly sensitive force probes has become established in the biophysical community, advancing our understanding of cellular processes and microbial behavior. The micropipette force sensor (MFS) technique relies on measurement of the forces acting on a force-calibrated, hollow glass micropipette by optically detecting its deflections. Read More

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http://www.nature.com/articles/s41596-018-0110-x
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http://dx.doi.org/10.1038/s41596-018-0110-xDOI Listing
February 2019
2 Reads

Publisher Correction: Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Nat Protoc 2019 Jan 29. Epub 2019 Jan 29.

The Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper. Read More

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http://dx.doi.org/10.1038/s41596-019-0138-6DOI Listing
January 2019

A procedure for identifying possible products in the assembly-disassembly-organization-reassembly (ADOR) synthesis of zeolites.

Nat Protoc 2019 Jan 25. Epub 2019 Jan 25.

School of Chemistry and EaStCHEM, University of St. Andrews, St. Andrews, UK.

High-silica zeolites, some of the most important and widely used catalysts in industry, have potential for application across a wide range of traditional and emerging technologies. The many structural topologies of zeolites have a variety of potential uses, so a strong drive to create new zeolites exists. Here, we present a protocol, the assembly-disassembly-organization-reassembly (ADOR) process, for a relatively new method of preparing these important solids. Read More

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http://dx.doi.org/10.1038/s41596-018-0114-6DOI Listing
January 2019

A surgical orthotopic approach for studying the invasive progression of human bladder cancer.

Nat Protoc 2019 Jan 25. Epub 2019 Jan 25.

Division of Urologic Oncology, Department of Urology, University of Michigan, Ann Arbor, MI, USA.

The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice. Read More

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http://dx.doi.org/10.1038/s41596-018-0112-8DOI Listing
January 2019
1 Read

Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.

Nat Protoc 2019 Feb;14(2):313-330

Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Targeted tandem mass spectrometry (LC-MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (C) and nitrogen-15 (N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. Read More

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http://www.nature.com/articles/s41596-018-0102-x
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http://dx.doi.org/10.1038/s41596-018-0102-xDOI Listing
February 2019
3 Reads

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells.

Nat Protoc 2019 Feb;14(2):616-638

Translational Imaging Center, Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA.

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. Read More

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http://www.nature.com/articles/s41596-018-0111-9
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http://dx.doi.org/10.1038/s41596-018-0111-9DOI Listing
February 2019
2 Reads

Generation of lung organoids from human pluripotent stem cells in vitro.

Nat Protoc 2019 Feb;14(2):518-540

Program in Cell and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA.

The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm-mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior-posterior and dorsal-ventral patterning, lung specification, lung budding, branching morphogenesis, and, finally, maturation. Here we describe a protocol that recapitulates several of these milestones in order to differentiate human pluripotent stem cells (hPSCs) into ventral-anterior foregut spheroids and further into two distinct types of organoids: human lung organoids and bud tip progenitor organoids. Read More

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http://dx.doi.org/10.1038/s41596-018-0104-8DOI Listing
February 2019
1 Read

Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.

Nat Protoc 2019 Feb;14(2):482-517

The Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. Read More

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http://dx.doi.org/10.1038/s41596-018-0103-9DOI Listing
February 2019
1 Read

High-fidelity probing of the structure and heterogeneity of extracellular vesicles by resonance-enhanced atomic force microscopy infrared spectroscopy.

Nat Protoc 2019 Feb;14(2):576-593

Faculty of Medicine and Health, Sydney Pharmacy School, The University of Sydney, Sydney, NSW, Australia.

Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. Read More

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http://www.nature.com/articles/s41596-018-0109-3
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http://dx.doi.org/10.1038/s41596-018-0109-3DOI Listing
February 2019
1 Read

Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

Nat Protoc 2019 Feb;14(2):415-440

Bioinformatics and Computational Biology Graduate Program, University of Minnesota Twin Cities, Minneapolis, MN, USA.

The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. Read More

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http://dx.doi.org/10.1038/s41596-018-0099-1DOI Listing
February 2019

Systemic AAV vectors for widespread and targeted gene delivery in rodents.

Nat Protoc 2019 Feb;14(2):379-414

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. Read More

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http://dx.doi.org/10.1038/s41596-018-0097-3DOI Listing
February 2019

In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells.

Nat Protoc 2019 Feb;14(2):350-378

Wellcome Trust Sanger Institute, Hinxton, UK.

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Read More

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http://dx.doi.org/10.1038/s41596-018-0096-4DOI Listing
February 2019

A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma.

Nat Protoc 2019 Feb;14(2):541-555

Edwin L. Steele Laboratories, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. Read More

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http://dx.doi.org/10.1038/s41596-018-0105-7DOI Listing
February 2019
17 Reads

Targeted intraspinal injections to assess therapies in rodent models of neurological disorders.

Nat Protoc 2019 Feb;14(2):331-349

Department of Neurology, University of Michigan, Ann Arbor, MI, USA.

Despite decades of research, pharmacological therapies for spinal cord motor pathologies are limited. Alternatives using macromolecular, viral, or cell-based therapies show early promise. However, introducing these substances into the spinal cord, past the blood-brain barrier, without causing injury is challenging. Read More

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http://www.nature.com/articles/s41596-018-0095-5
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http://dx.doi.org/10.1038/s41596-018-0095-5DOI Listing
February 2019
7 Reads

Life cycle maintenance and drug-sensitivity assays for early drug discovery in Schistosoma mansoni.

Nat Protoc 2019 Feb;14(2):461-481

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland.

Drug discovery for schistosomiasis is still limited to a handful of academic laboratories worldwide, with only a few novel antischistosomal lead compounds being actively researched. Despite recent international mobilization against the disease to stimulate and promote antischistosomal drug discovery, setting up a drug-screening flow with schistosome parasites remains challenging. Whereas numerous different protocols to obtain and cultivate schistosomes have been published, those describing the drug-screening process are scarce, and none gather together parasite cultivation and early drug discovery procedures. Read More

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http://dx.doi.org/10.1038/s41596-018-0101-yDOI Listing
February 2019

Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo.

Nat Protoc 2019 Feb;14(2):556-575

Max Planck Institute for Brain Research, Frankfurt, Germany.

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). Read More

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http://dx.doi.org/10.1038/s41596-018-0106-6DOI Listing
February 2019

Cell-type-specific quantification of protein synthesis in vivo.

Nat Protoc 2019 Feb;14(2):441-460

Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center, University of California San Diego, La Jolla, CA, USA.

Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Read More

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http://dx.doi.org/10.1038/s41596-018-0100-zDOI Listing
February 2019

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

Nat Protoc 2019 Jan;14(1):283-312

Center for Integrated Protein Science Munich (CiPSM), Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e. Read More

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http://www.nature.com/articles/s41596-018-0094-6
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http://dx.doi.org/10.1038/s41596-018-0094-6DOI Listing
January 2019
9 Reads

Quantitative cross-linking/mass spectrometry to elucidate structural changes in proteins and their complexes.

Nat Protoc 2019 Jan;14(1):171-201

Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.

Quantitative cross-linking/mass spectrometry (QCLMS/QXL-MS) probes structural changes of proteins in solution. This method has revealed induced conformational changes, composition shifts in conformational ensembles and changes in protein interactions. It illuminates different structural states of proteins or protein complexes by comparing which residue pairs can be cross-linked in these states. Read More

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http://dx.doi.org/10.1038/s41596-018-0089-3DOI Listing
January 2019

CRISPR-Cas9 genome engineering of primary CD4 T cells for the interrogation of HIV-host factor interactions.

Nat Protoc 2019 Jan;14(1):1-27

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.

CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4 T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4 T cells by nucleofection. Read More

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http://dx.doi.org/10.1038/s41596-018-0069-7DOI Listing
January 2019
5 Reads

Application of anisotropic NMR parameters to the confirmation of molecular structure.

Nat Protoc 2019 Jan;14(1):217-247

Structure Elucidation Group, Process and Analytical Research and Development, Merck Research Laboratories, Rahway, NJ, USA.

The use of anisotropic NMR data, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), has emerged as a powerful technique for structural characterization of organic small molecules. RDCs typically report the relative orientations of different H-C bonds; RCSAs report the relative orientations of different carbon chemical shielding tensors and hence are more useful for proton-deficient molecules. This information is complementary to that obtained from conventional NMR data such as J couplings, isotropic chemical shifts, and nuclear Overhauser effects (NOEs)/rotational frame nuclear Overhauser effects (ROEs). Read More

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http://dx.doi.org/10.1038/s41596-018-0091-9DOI Listing
January 2019
1 Read

Genome-wide mapping of nucleotide excision repair with XR-seq.

Nat Protoc 2019 Jan;14(1):248-282

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA.

Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed excision repair sequencing (XR-seq) to study nucleotide excision repair of DNA adducts in humans, mice, Arabidopsis thaliana, yeast and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction, are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. Read More

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http://www.nature.com/articles/s41596-018-0093-7
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http://dx.doi.org/10.1038/s41596-018-0093-7DOI Listing
January 2019
19 Reads

Resolution of the DNA methylation state of single CpG dyads using in silico strand annealing and WGBS data.

Nat Protoc 2019 Jan;14(1):202-216

Department of Biology, Emory University, Atlanta, GA, USA.

Whole-genome bisulfite sequencing (WGBS) has been widely used to quantify cytosine DNA methylation frequency in an expanding array of cell and tissue types. Because of the denaturing conditions used, this method ultimately leads to the measurement of methylation frequencies at single cytosines. Hence, the methylation frequency of CpG dyads (two complementary CG dinucleotides) can be only indirectly inferred by overlaying the methylation frequency of two cytosines measured independently. Read More

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http://dx.doi.org/10.1038/s41596-018-0090-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311134PMC
January 2019

Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Nat Protoc 2019 Jan;14(1):153-170

Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Cambridge, UK.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. Read More

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http://www.nature.com/articles/s41596-018-0087-5
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http://dx.doi.org/10.1038/s41596-018-0087-5DOI Listing
January 2019
17 Reads

A comprehensive pipeline for translational top-down proteomics from a single blood draw.

Nat Protoc 2019 Jan;14(1):119-152

Departments of Chemistry and of Molecular Biosciences, Northwestern University, Evanston, IL, USA.

Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. Read More

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http://dx.doi.org/10.1038/s41596-018-0085-7DOI Listing
January 2019
1 Read

Author Correction: Efficient genetic engineering of human intestinal organoids using electroporation.

Nat Protoc 2018 Dec 5. Epub 2018 Dec 5.

Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.

The version of this paper originally published shows incorrect units for two plasmid concentrations. In the "Reagent Setup" section, the instructions for sgRNA-Cas9 plasmid should read "Adjust the concentration of each plasmid to 1 μg μl," rather than "to 1 μg ml." Similarly, all concentrations in the tables in Steps 49A, 49C, and 49D should be in μg μl instead of μg ml. Read More

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http://dx.doi.org/10.1038/s41596-018-0107-5DOI Listing
December 2018
1 Read

Publisher Correction: Acute and rapid degradation of endogenous proteins by Trim-Away.

Nat Protoc 2018 Nov 30. Epub 2018 Nov 30.

Laboratory of Molecular Biology, Medical Research Council, Cambridge, UK.

In the version of this paper originally published, the present address of W.A. McEwan was accidentally omitted. Read More

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http://dx.doi.org/10.1038/s41596-018-0092-8DOI Listing
November 2018

Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy.

Nat Protoc 2019 Jan;14(1):100-118

The Astbury Biostructure Laboratory, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.

The dramatic growth in the use of cryo-electron microscopy (cryo-EM) to generate high-resolution structures of macromolecular complexes has changed the landscape of structural biology. The majority of structures deposited in the Electron Microscopy Data Bank (EMDB) at higher than 4-Å resolution were collected on Titan Krios microscopes. Although the pipeline for single-particle data collection is becoming routine, there is much variation in how sessions are set up. Read More

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http://dx.doi.org/10.1038/s41596-018-0084-8DOI Listing
January 2019

Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells.

Nat Protoc 2019 Jan;14(1):51-67

Department of Molecular Biology, University of Geneva, Geneva, Switzerland.

A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of mapping DNA replication origins in higher eukaryotes rely principally on fractionation of DNA fragments based on their size and, optionally, on the presence of ribonucleotides at their 5' end. Here, we describe a protocol for EdUseq-HU, a method for mapping early S-phase replication origins. Read More

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http://www.nature.com/articles/s41596-018-0081-y
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http://dx.doi.org/10.1038/s41596-018-0081-yDOI Listing
January 2019
10 Reads

Generation of human antral and fundic gastric organoids from pluripotent stem cells.

Nat Protoc 2019 Jan;14(1):28-50

Center for Stem Cell and Organoid Medicine, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Read More

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http://dx.doi.org/10.1038/s41596-018-0080-zDOI Listing
January 2019

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

Nat Protoc 2019 Jan;14(1):86-99

Department of Chemistry, University of Cambridge, Cambridge, UK.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. Read More

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http://dx.doi.org/10.1038/s41596-018-0083-9DOI Listing
January 2019
9 Reads

Single-pot, solid-phase-enhanced sample preparation for proteomics experiments.

Nat Protoc 2019 Jan;14(1):68-85

Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

A critical step in proteomics analysis is the optimal extraction and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technology that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-preparation (SP3) technology is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic analysis. Read More

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http://www.nature.com/articles/s41596-018-0082-x
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http://dx.doi.org/10.1038/s41596-018-0082-xDOI Listing
January 2019
16 Reads

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Nat Protoc 2018 Dec;13(12):2991-3017

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. Read More

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http://www.nature.com/articles/s41596-018-0075-9
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http://dx.doi.org/10.1038/s41596-018-0075-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364805PMC
December 2018
15 Reads

Modular approach for resolving and mapping complex neural and other cellular structures and their associated deformation fields in three dimensions.

Nat Protoc 2018 Dec;13(12):3042-3064

Department of Mechanical Engineering, University Wisconsin-Madison, Madison, WI, USA.

Understanding the biological implications of cellular mechanotransduction, especially in the context of pathogenesis, requires the accurate resolution of material deformation and strain fields surrounding the cells. This is particularly challenging for cells displaying branched, 3D architectures. Here, we provide a modular approach for 3D image segmentation and strain mapping of topologically complex structures. Read More

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http://dx.doi.org/10.1038/s41596-018-0077-7DOI Listing
December 2018
10 Reads

Introducing our Tutorials.

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Nat Protoc 2018 Dec;13(12):2741

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http://dx.doi.org/10.1038/s41596-018-0078-6DOI Listing
December 2018

Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM.

Nat Protoc 2018 Dec;13(12):2890-2907

Materials Science Factory, Instituto de Ciencia de Materiales de Madrid (ICMM), CSIC, Madrid, Spain.

Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. Read More

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http://www.nature.com/articles/s41596-018-0070-1
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http://dx.doi.org/10.1038/s41596-018-0070-1DOI Listing
December 2018
16 Reads

Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies.

Nat Protoc 2018 Dec;13(12):2742-2757

CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.

Single-cell RNA sequencing is at the forefront of high-resolution phenotyping experiments for complex samples. Although this methodology requires specialized equipment and expertise, it is now widely applied in research. However, it is challenging to create broadly applicable experimental designs because each experiment requires the user to make informed decisions about sample preparation, RNA sequencing and data analysis. Read More

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http://www.nature.com/articles/s41596-018-0073-y
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http://dx.doi.org/10.1038/s41596-018-0073-yDOI Listing
December 2018
29 Reads

Mitigating head motion artifact in functional connectivity MRI.

Nat Protoc 2018 Dec;13(12):2801-2826

Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA.

Participant motion during functional magnetic resonance image (fMRI) acquisition produces spurious signal fluctuations that can confound measures of functional connectivity. Without mitigation, motion artifact can bias statistical inferences about relationships between connectivity and individual differences. To counteract motion artifact, this protocol describes the implementation of a validated, high-performance denoising strategy that combines a set of model features, including physiological signals, motion estimates, and mathematical expansions, to target both widespread and focal effects of subject movement. Read More

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http://www.nature.com/articles/s41596-018-0065-y
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http://dx.doi.org/10.1038/s41596-018-0065-yDOI Listing
December 2018
21 Reads

Efficient and robust proteome-wide approaches for cross-linking mass spectrometry.

Nat Protoc 2018 Dec;13(12):2964-2990

Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands.

Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. Read More

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http://www.nature.com/articles/s41596-018-0074-x
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http://dx.doi.org/10.1038/s41596-018-0074-xDOI Listing
December 2018
12 Reads

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research.

Nat Protoc 2018 Dec;13(12):2944-2963

Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, Australia.

'Speed breeding' (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants' daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber-based SB approaches with supporting data from experimentation with several crops. Read More

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http://www.nature.com/articles/s41596-018-0072-z
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http://dx.doi.org/10.1038/s41596-018-0072-zDOI Listing
December 2018
78 Reads

Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Nat Protoc 2018 Dec;13(12):2908-2943

The Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. Read More

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http://www.nature.com/articles/s41596-018-0071-0
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http://dx.doi.org/10.1038/s41596-018-0071-0DOI Listing
December 2018
9 Reads