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Quantitative cross-linking/mass spectrometry is used to discover protein structural changes (e.g., in protein interactions). The cross-linker bis(sulfosuccinimidyl)suberate is used for both label-free and isotope-labeled workflows in this protocol.
In addition to canonical nucleotides, DNA contains various modified bases that contribute to the developmental and disease state of the organism. UHPLC–MS/MS with isotopically labeled standards is used in this protocol to quantify modified bases.
In this protocol, the authors describe how to design, synthesize, and deliver CRISPR–Cas9 RNPs to primary CD4+ T cells for targeted gene knockout. They then show how the edited cells can be used for the analysis of host factors in HIV replication.
Liu et al. expand the toolset available for NMR characterization of organic compounds by providing a protocol for the generation and analysis of anisotropic NMR data (residual dipolar couplings and residual chemical shift anisotropies).
Nucleotide excision repair is a conserved pathway to resolve bulky DNA adducts caused by different mutagens. XR-seq generates genome-wide repair maps of DNA damage across a range of organisms.
iSA uses a series of computational steps to resolve the methylation status of individual Cs in a CpG dyad. This versatile approach can be applied downstream of available whole-genome bisulfite sequencing data.
This protocol describes DIVA, a genome-wide method to compare chromatin accessibility between cell types. Decompacted chromatin is more susceptible to lentivirus integration, which is captured by large-scale sequencing of virus–genome junctions.
In top-down proteomics, intact proteins are analyzed by mass spectrometry. Toby et al. describe an approach for analyzing peripheral blood mononuclear cells by top-down Fourier-transform MS, with data analysis using ProSight Lite and TDPortal.
This protocol describes a pipeline for data collection, pre-processing and on-the-fly analysis for single-particle cryo-electron microscopy using EPU software and two direct electron detectors: the Thermo Fisher Scientific Falcon 3 and the Gatan K2.
This protocol describes high-throughput sequencing strategies for mapping early-firing replication origins and fork movement. Both procedures rely on EdU labeling of nascent DNA and the use of hydroxyurea to limit fork progression in synchronized cells.
In this protocol, growth factors, growth factor inhibitors, and small molecules are used to direct the stepwise differentiation of human pluripotent stem cells into antral and fundic gastric organoids containing functional gastric cells.
Site-selective modification of antibodies is useful for therapeutic and imaging applications. This protocol is an alternative to maleimide labeling in which cysteine reacts selectively and irreversibly with functionalized carbonylacrylic reagents.
This protocol describes a single-pot, solid-phase-enhanced sample-preparation (SP3) method for rapid, robust, and efficient processing of protein samples for proteomic analysis.
3D image segmentation and strain mapping are applied to topologically complex structures. As an example, we present the resolution of 3D strains on a culture containing neurons, astrocytes, and neural progenitors undergoing an in vitro injury event.
This protocol describes a suite of lentiviral transfer plasmids that can be used for high-yield, time- and cost-efficient, and constitutive or inducible production of soluble and membrane proteins in mammalian cell lines.
Cross-linking of amino acids in close proximity on protein surfaces, followed by mass spectrometry, provides useful structural information. This protocol for using XlinkX can be applied for analysis of cultured cell samples or purified complexes.
Ciric et al. describe a protocol for the removal of motion artifacts from functional MRI data. They introduce a software package that implements common denoising protocols and provides tools for assessing the efficacy of denoising.
This protocol describes procedures for speed-breeding approaches using growth cabinets and LED-supplemented glasshouses. The approaches can be used to accelerate crop research and are compatible with a wide variety of crops.
This protocol describes how to generate high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. The procedure covers sample preparation, bimodal AFM setup and calibration, and data acquisition and processing.
This protocol describes strategies for targeted genome modification in axolotls. Eggs are injected with a CAS9–gRNA ribonucleoprotein complex, which allows for efficient generation of knockout and knock-in animals and immediate phenotypic analysis.