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This protocol describes a platform for directly converting human mid-gestation lineage-committed amniotic fluid-derived cells into a stable and expandable population of vascular endothelial cells over a 3-week period without using pluripotency factors.
The Fraser laboratory provides a protocol for performing Hi-C on single cells. The technique is an important step towards understanding cellular variability in genome organization and chromosome conformation.
Carbon-hydrogen bond functionalization is a desirable strategy for the selective derivatization of organic molecules. This protocol describes the direct silylation of aromatic heterocycles using an inexpensive and abundant catalyst: potassium tert-butoxide.
Targeted RNA directional sequencing (TARDIS) combines targeted RNA capture (using DNA traps) with strand-specific sequencing to enable discovery of rare transcripts, including long and short noncoding RNAs.
Human noroviruses are a leading cause of foodborne disease. Here the authors facilitate the study of these viruses with protocols for culturing them in human B cells and assessing bacterial stimulation of B cell infection and viral attachment to B cells.
This protocol describes how to prepare thick sections of mouse skeletal tissue for high-resolution 3D confocal imaging. Samples prepared in this way retain both fluorescence activity and antigenicity, allowing simultaneous imaging of multiple markers.
This protocol describes how to fix, embed, clear and stain excised organs or whole organisms to create optically transparent samples. This versatile protocol is able to process a wide range of sample types for high-resolution imaging.
Here, the authors describe genetically engineering the Pseudomonas genome by two-step allelic exchange. Suicide vector-encoded alleles are used to generate mutations by homologous recombination at the single base pair level.
In this protocol, the authors describe methods for the direct delivery of ZFN, TALEN and Cas9 nuclease proteins into cells for efficient and targeted genome editing.
This protocol describes a modular approach to modify existing genetically engineered mouse models (GEMMs) by re-derivation of embryonic stem cells (ESCs) and subsequent modification of these cells by recombinase-mediated transgene insertion.
Pu-seq (polymerase usage sequencing) is an approach for identifying ribonucleotides by high-throughput sequencing to allow the mapping of replicative polymerase usage genome wide. This has been used to define origin efficiencies and replication timing in yeast.
Changes in the levels of cellular thiols (e.g., glutathione) are linked to many diseases. This protocol is for the synthesis of CPDSA, a fluorescent turn-on glutathione probe with near-infrared emission. In-cell and in vivo assays are also described.
The micro-EROD assay is a high-throughput screening method used to assess the CYP1A-inducing potential of dioxins and dioxin-like chemicals in rat hepatoma cells.
Rat or human lung samples are enzymatically digested, and CD31-expressing cells are positively selected using magnetic-activated cell sorting before plating in endothelial-specific growth conditions.
This protocol describes how to perform CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis), a simple and efficient method for organ clearing, imaging by light-sheet microscopy, and quantitative imaging analysis.
When an electric field is applied across a membrane containing nanopores, it induces an ion current that alters upon ligand binding. Single-walled carbon nanotubes form nanopores that can be used as sensors; specificity is a function of pore size.
In SHAPE-MaP, reverse transcriptase–induced mutations at SHAPE-modified RNA nucleotides are detected by high-throughput sequencing. ShapeMapper converts sequence data to mutational profiles (MaPs), which can be used by SuperFold for RNA structure modeling.
The authors describe methods for the directed evolution of artificial endonuclease and ligase enzymes by X-SELEX, from diverse repertoires of synthetic genetic polymers (XNAzymes). The protocol has been applied to four different XNA chemistries and three different reactions, and it is, in principle, applicable to many more.