Volume 14 Issue 11, November 2019

Microvessels are isolated from mouse brain cortex, minimizing cell activation and yielding microvessel fragments with consistent populations of discrete blood–brain barrier components that retain RNA integrity and protein post-translational modifications.

Self-organizing 3D human blood vessel organoids are generated by mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I–Matrigel matrix.

Fusing the SNAP-tag to a disease-specific protein of interest allows its directed functionalization with a synthetic benzylguanine-modified diagnostic or therapeutic label in a 1:1 stoichiometry. This protocol describes how to produce, conjugate and test the activity of the corresponding combination products.

This protocol uses a microfluidic device and microscopy to detect mutations occurring as a consequence of DNA replication and assess their effects on fitness in Escherichia coli single cells.

BACMMAN software is used to automate image analysis of high-throughput 2D or 3D time-series images from experiments using the ‘mother machine’, a microfluidic device that allows growth and division of single bacterial cells to be followed.

This protocol describes SpiderMass, a platform for in vivo and real-time mass spectrometry analysis. The procedure describes system setup and calibration; analysis of cell culture, tissue section and in vivo skin samples; and data processing.

This protocol combines hydrogen–deuterium exchange mass spectrometry (HDX-MS) of membrane proteins in nanodiscs with molecular dynamics (MD) simulations to identify lipid–protein interactions that modulate conformational changes.

This protocol describes the targeted viral infection of single cells or small groups of cells by magnetically guided virus stamping, allowing for controlled genetic engineering, exemplified here by stamping cultured neurons with adeno-associated viruses.

tRNA modifications are important for normal and disease states, but methods to study them are lacking. This protocol relies on chemical cleavage at 7-methylguanosine (m⁷G) sites, followed by sequencing, to precisely map these modifications genome-wide.

iMARGI (in situ mapping of RNA–genome interactome) is a proximity ligation method for global profiling of chromatin-associated RNAs. A linker sequence bridges DNA and RNA in physical proximity, permitting sequencing library preparation and mapping of DNA–RNA contacts.