Volume 15 Issue 2, February 2020

Oxygen plays key roles in bioenergetics, metabolism, signaling pathways and developmental biology. This protocol describes how to perform quantitative oxygen flux measurements on cells, ex vivo tissues and various model animals in vivo.

This native mass spectrometry approach allows rapid, in-depth characterization of protein structure and function directly from crude samples, bypassing the need for prior purification.

Here, the authors present standardized computational pipelines tailored specifically to the analysis of cancer genome sequencing data from mice. The protocol enables detection of single-nucleotide variants, indels, copy-number variations, loss of heterozygosity and complex rearrangements such as those of chromothripsis.

This protocol manipulates the hydrophobicity of DNA and tailors its conformation at the water–oil interface on an organogel surface. Measuring the sliding speed or the critical sliding angle offers a droplet motion-based biosensing platform for the visual detection of small molecules, nucleic acids and proteins.

In this protocol, the authors describe a method to fine-tune gene expression in plants by editing endogenous upstream ORFs with the CRISPR–Cas9 system to prevent their inhibition of the translation of primary ORFs. This protocol yields transgene-free uorf mutant offspring.

This protocol presents experimental and computational procedures for unbiased analysis of multi-way chromatin contacts at a selected genomic site. Multi-way contacts are captured by proximity ligation and detected using long-read sequencing.

This protocol describes FLOW-MAP, a graph-based algorithm for visualizing cellular trajectories in single-cell time course datasets. The R package can be operated via its GUI or using text-based commands.

iPSC-derived motor neurons and skeletal muscle cells are co-cultured to establish a model of the human neuromuscular junction (NMJ) within a microfluidic device, which facilitates assessment of axonal outgrowth, NMJ formation and muscle maturation.

This protocol describes a screening pipeline to select red fluorescent proteins (FPs) with optimized parameters from a mutant FP library. FPs are screened for fluorescence lifetime, cellular brightness, maturation efficiency, and photostability.

This work compares several ML and calibration algorithms for classifying tumor DNA methylation profiles. The resulting protocol provides workflows for selecting, training and calibrating ML algorithms to generate well-calibrated multiclass probability estimates.

Record-seq is a technology that enables transcriptional recording by CRISPR spacer acquisition from RNA. Transcriptomes are recovered using SENECA, a method that selectively amplifies expanded CRISPR arrays, followed by deep sequencing.

A series of techniques (echocardiography, novel object recognition, grip strength, rotarod, glucose and insulin tolerance tests, body composition, and energy expenditure) are used to assess the health of mice.

Here, the authors describe how to download, modify and use PFIboxes—3D-printed fluorescence imaging boxes—to assay bacterial gene expression over time in response to stress using a fluorescent reporter library.

4-thiouridine (4SU) labeling of nascent transcripts enables high-resolution genome-wide profiling of newly transcribed RNAs in mammalian cells. In combination with transient transcription inhibition, RNA polymerase II elongation speeds can be measured.

Functional mouse hematopoietic stem cells (HSCs) are expanded 236- to 899-fold ex vivo using a fully defined albumin-free culture system. Clonal analysis of HSC heterogeneity and HSC transplantation are also described.

This analysis of chemical reaction mechanisms is based on the activation strain model of reactivity. The PyFrag 2019 program is used to perform activation strain and energy decomposition analysis on each point along the energy profile.

This protocol describes how to detect immature neurons in humans from post-mortem samples, indicating that neurogenesis has recently taken place. Sample quality plus appropriate tissue processing and histological procedures are all required.

This protocol describes a defined, xeno-free culture system to support long-term ex vivo expansion of functional human epidermal keratinocytes.