Volume 17 Issue 2, February 2022

The authors describe how to establish dissociated cultures of neurons, astrocytes, microglia, pericytes, and brain endothelial and neural precursor cells, as well as explant cultures of the leptomeninges, cortical slice cultures and brain tumor cells.

The authors present a protocol that enables isolation of growth cones and paired somata from any neuronal subtype of the CNS that can be fluorescently labeled by fluid shear forces, ultracentrifugation, fluorescent small particle sorting and FACS.

Bacterial genome-wide gene fitness is assessed by CRISPRi-seq. The procedure includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays, sequencing and read analysis fitness quantification.

Star-shaped DNA nanostructures are designed to inhibit dengue virus in vitro. The ‘DNA star’ acts as a template to display ten virus-binding aptamers with precise spatial patterning to mirror the virus antigens, achieving high viral binding avidity.

Mirkin and colleagues outline the synthesis of protein spherical nucleic acids (ProSNAs), which constitute a versatile plug-and-play platform that uses protein functionalization with a dense nucleic acid corona to enable intracellular delivery both in vitro and in vivo.

The electrochemical lithium ion intercalation-based exfoliation of mono- or few-layer transition metal dichalcogenides nanosheets described here results in materials that can be used in diverse applications, e.g., biosensing and catalysis.

IBEX (iterative bleaching extends multiplexity) is an iterative immunolabeling and chemical bleaching method that enables highly multiplexed imaging in diverse tissues.

KAS-seq is an N₃-kethoxal–assisted single-stranded DNA sequencing approach that allows rapid, sensitive and genome-wide mapping of single-stranded DNA produced in situ using as few as 1,000 cells or animal tissues.

Cryo-electron tomography with subtomogram averaging is useful for the structural analysis of heterogeneous protein complexes. emClarity is graphics processing unit-accelerated image-processing software for subtomogram averaging and classification at high resolution.

Capture-C detects DNA regions in close proximity in the nucleus, relying on oligonucleotide probes to enrich fragments of interest. This protocol describes several variations of this modular approach and a computational pipeline.

In this protocol, CRISPR perturbations are introduced in cells using lentiviral libraries and read out using in situ sequencing, coupling high-throughput pooled screening with phenotypic image readouts in live or fixed cells.

iPSC-derived macrophages (iPSC-Mac) are mass-produced in a scalable suspension culture on an orbital shaker or in a stirred-tank bioreactor, and can be harvested at weekly intervals for several months.

This protocol describes a strategy to stabilize integral membrane proteins by fusing their two termini to a self-assembling coupler protein, which also enables crystallization and facilitates every step in obtaining high-resolution structures.