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Human gastric organoids from pluripotent stem cells
Human-pluripotent-stem-cell-derived fundic gastric organoids and antral organoids at day 20 of culture. The images show immunofluorescent staining of the organoids for the markers GATA4 (green), PDX1 (red) and CDH1/E-cadherin (white), as well as the nuclear stain DAPI (blue).
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.
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.
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.
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.
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 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.
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 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.
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.
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.
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.
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.