Volume 16 Issue 2, February 2021

In this Protocol Extension, Lancaster et al. describe a modified version of their original protocol (published in 2014) that can be used to reliably generate cerebral organoids of a telencephalic identity and maintain long-term viability for later stages of neural development, including axon outgrowth and neuronal maturation.

This Protocol Extension presents recombinant extracellular vesicles as reference materials for method development and standardization. It details their characterization and detection in spiked samples by fluorescence, nucleic acid and protein measurements.

This protocol describes a microfluidic platform for high-throughput sorting of individual cells from microbial communities. Metabolically active cells are labeled using D₂O or ¹³C, selected by Raman imaging, and sorted on-chip with optical tweezers.

This protocol describes how to assemble a 4Pi single-molecule switching super-resolution microscope. Detailed instructions for beam-path alignment, testing, application to cellular samples and troubleshooting are provided.

OCTAD is a software pipeline for finding drugs that alter gene expression in such a way that they are likely to reverse the expression pattern of the disease. This protocol describes how to use both the web portal and the desktop version of OCTAD.

This protocol describes VAMPIRE, an unsupervised machine-learning approach that can be used to quantify and categorize cellular morphology from fluorescence or bright-field images of cells grown in 2D, 3D and tissue slices.

A neonatal mouse model of pressure overload by transverse aortic constriction (nTAC) is described. Mice fully adapt following nTAC on postnatal day 1, but if nTAC is applied on day 7, a maladaptive response occurs that is similar to that seen in adult mice following TAC.

This protocol describes how to follow the behavior of immune cells by imaging ex vivo cultured human or mouse skin biopsies following labeling with antibody or nanobody reagents against specific cell surface markers.

The authors discuss experimental design considerations and describe a computational pipeline to reveal the synergistic and additive effects of combinatorial perturbations on gene expression measured by RNA sequencing.

This protocol describes a workflow that combines laser-capture microdissection with low-input genome sequencing, while circumventing the use of whole-genome amplification, for accurate detection of somatic mutations in non-neoplastic tissues.

Malanchi and colleagues describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein.

A new protocol for solid-state NMR of soluble and membrane proteins in E. coli, in both whole cells and isolated membrane fractions. The procedure describes conventional ¹³C/¹⁵N ssNMR as well as sensitivity-enhanced DNP-ssNMR and ¹H-detected ssNMR.

Takebe et al. describe a protocol for the continuous patterning of hepatic, biliary and pancreatic structures from a 3D culture of human pluripotent stem cells.

This protocol describes the construction of spinning microfluidics platforms for facile production of perfusable hydrogel microtubes of various sizes and shapes. The microtubes can be loaded with cells to create biomimetic vascular channels.

Reversible protection of the primary face of cyclodextrins by silylation is a very popular strategy for modification of the secondary rim. This protocol describes how to prepare these important intermediates in high yield and purity.

Cultivating native bacteria from fresh plant roots is essential for understanding their interaction with the host plant. This protocol describes their isolation and accurate taxonomical identification using two-sided barcode polymerase chain reaction and Illumina sequencing.

Mancuso and De Strooper and colleagues describe MIGRATE (microglia in vitro generation refined for advanced transplantation experiments, a combined in vitro differentiation and in vivo xenotransplantation protocol for studying human microglia transplanted into mouse brain.

This protocol describes a genome-wide approach for ultrasensitive and quantitative detection of DNA double-strand breaks (DSBs) that relies on encapsulating cells in agarose beads and labeling breaks with biotinylated adapters.

This protocol describes how to perform combined protein isoform detection with nucleic acid analysis from the same individual mouse embryo or blastomere using fractionation polyacrylamide gel electrophoresis (fPAGE).

This protocol for base editing in cultured mammalian cells provides guidelines for choosing target sites, appropriate base editor variants and delivery strategies, as well as detailing the computational analysis of base-editing outcomes using CRISPResso2.

The authors describe hardware setup and experimental workflows for collecting and analyzing the biotic and abiotic environmental exposome at the individual level.

Optimization of chemical reactions can be facilitated by techniques that enable experiments to be set up in parallel. In this work, 96 Pd–catalyzed cross-coupling experiments are performed and analyzed in parallel with commonly available equipment.

This protocol describes a cell-free method for experimentally identifying genome-wide off-target sites of CRISPR nucleases and deaminases through in vitro digestion of genomic DNA or chromatin followed by whole-genome sequencing.

This protocol describes an approach to quantifying DNA replication dynamics (initiation and termination frequencies and origin firing efficiencies) at defined genomic loci in asynchronously growing cells.

This protocol describes procedures for high-throughput analysis of trigenic interactions in yeast. Triple-mutant strains generated in a series of automated replica-pinning steps are grown on agar plates as individual colonies, and interactions are quantified with the trigenic synthetic genetic array scoring method.

A protocol for implementing Brillouin microscopy to study biological materials. The procedure contains instructions for integrating an add-on Brillouin module with an existing confocal microscope as well as for its calibration and use in data collection and processing.

The authors describe a modular pipeline to detect aberrant gene expression events (expression level, splicing and mono-allelic expression) from patient RNA sequencing data, which can complement DNA-based diagnosis by enhancing the functional interpretation of variants.

This protocol describes how to perform near-infrared spectroscopy and imaging of connective tissues. Detailed guidelines are provided for sample preparation, spectral acquisition and data pre-processing and analysis, with example applications.