Nature Protocols最新文献

Cartilage is an essential component of the vertebrate skeleton, providing biomechanical support via its extracellular matrix composition. However, in many mammals, including humans and mice, numerous head, neck and chest cartilages produce little extracellular matrix and, instead, contain many large intracellular lipid vacuoles, which determine tissue size, shape and biomechanics. Such cartilages, termed lipocartilages, are made of individual cells called lipochondrocytes with distinct gene expression, lipid composition and metabolism. Lipochondrocytes significantly influence tissue-level physiology, regenerative potential and aging of skeletal elements. Here we provide a step-by-step protocol for the isolation of lipocartilage from mouse ear and the purification of its lipochondrocytes. We include instructions on how to microdissect ear lipocartilage for the purposes of lipid staining, wholemount imaging, morphometric analyses and biomechanical assays. Furthermore, we include a guide for the efficient dissociation of lipocartilages and the purification of individual lipochondrocytes by means of lipid-based buoyancy or cell sorting following fluorescent staining with neutral lipid dyes. With adequate dissection tools and sufficient practice, a researcher can cleanly isolate mouse ear lipocartilage within 20 min and purify lipochondrocytes within 4 h. Tissue biomechanics can be assayed by tensile testing within 30 min per sample. Although the protocol has only been validated in mice, it might be possible to adapt it for larger mammals, but modifications would probably be necessary, as lipocartilage is thicker. These guidelines will serve as a standard for future experiments on lipocartilage and have applications in the fields of developmental biology, bioengineering and metabolism.

Neural activity data can be associated with behavioral and physiological variables by analyzing their changes in the temporal domain. However, such relationships are often difficult to quantify and test, requiring advanced computational modeling approaches. Here, we provide a protocol for the statistical analysis of brain dynamics and for testing their associations with behavioral, physiological and other non-imaging variables. The protocol is based on an open-source Python package built on a generalization of the hidden Markov model (HMM)-the Gaussian-linear HMM-and supports multiple experimental modalities, including task-based and resting-state studies, often used to explore a wide range of questions in neuroscience and mental health. Our toolbox is available as both a Python library and a graphical interface, so it can be used by researchers with or without programming experience. Statistical inference is performed by using permutation-based methods and structured Monte Carlo resampling, and the framework can easily handle confounding variables, multiple testing corrections and hierarchical relationships within the data, among other features. The package includes tools developed to facilitate the intuitive visualization of statistical results, along with comprehensive documentation and step-by-step tutorials for data interpretation. Overall, the protocol covers the full workflow for the statistical analysis of functional neural data and their temporal dynamics.

Genomic loci positioning by sequencing (GPSeq) is a genome-wide method for mapping the radial organization of the genome in the nucleus of eukaryotic cells. GPSeq relies on in situ digestion of chromatin with a restriction enzyme that gradually diffuses inward from the nuclear periphery, followed by ligation of sequencing adapters to the digested restriction enzyme sites and library preparation for high-throughput sequencing. In parallel, ligation of labeled imaging adapters to the digested restriction enzyme recognition sites enables monitoring of the progression of radial digestion by fluorescence microscopy, providing an essential internal quality control before proceeding with sequencing. By comparing samples in which chromatin has been digested for increasing time intervals, a GPSeq score is calculated for every genomic bin into which the genome is arbitrarily divided, and genome-wide radial maps are generated with a resolution as high as 25 kb. These maps allow exploration of the radial distribution of (epi)genomic features, gene expression levels, mutational landscapes, and genomic profiles of DNA damage, when integrated with other omic data. Here, we present a detailed step-by-step protocol for performing GPSeq and preprocessing GPSeq data. The entire protocol requires ~2 weeks from the start of sample preparation to having ready-to-sequence libraries and intermediate levels of expertise in molecular biology, genomics and microscopy.

High-resolution mapping of active RNA polymerase II transcription initiation provides a dynamic view of gene expression and reveals the entire spectrum of RNA transcripts-from stable mRNAs to transient enhancer RNAs-which is critical for understanding gene regulation, deciphering transcriptional programs and defining regulatory element function. Here we present a detailed protocol for capped small RNA sequencing (csRNA-seq). Starting with total RNA, which can be readily isolated from fresh, frozen or fixed cells, tissues or patient samples, csRNA-seq selectively enriches for actively initiating 5'-capped RNA polymerase II transcripts. This approach captures both initiating stable protein-coding RNAs and non-coding RNAs, as well as rapidly degraded, transient transcripts such as enhancer or promoter divergent RNAs, providing a comprehensive snapshot of active cis-regulatory elements and facilitating the study of underlying regulatory mechanisms with high sensitivity. The protocol involves small RNA isolation, 5'-capped RNA enrichment and library generation, followed by sequencing. Key advantages of csRNA-seq over other nascent RNA-seq methods include (i) decoupling of sample collection and processing, (ii) broad compatibility with diverse eukaryotic sample types and organisms, (iii) high-resolution data defining active regulatory elements and their properties and (iv) scalability. Importantly, purified RNA is non-infectious and can be isolated from inactivated samples, including clinical or pathogenic specimens, allowing safe transport and analysis under standard laboratory conditions. This protocol empowers researchers with minimal experience in nascent transcriptomics to study gene regulation, cis-regulatory elements and transcription dynamics.

Uneven translation rates resulting from mRNA context, tRNA abundance, nascent amino acid sequence or various external factors play a key role in controlling the expression level and folding of the proteome. Inverse toeprinting coupled to next-generation sequencing (iTP-seq) is a scalable in vitro method for characterizing bacterial translation landscapes, complementary to ribosome profiling (Ribo-seq), a widely used method for determining transcriptome-wide protein synthesis rates in vivo. In iTP-seq, ribosome-protected mRNA fragments known as inverse toeprints are generated by using RNase R, a highly processive 3' to 5' RNA exonuclease. Deep sequencing of these fragments reveals the position of the leading ribosome on each mRNA with codon resolution, as well as the full upstream coding regions translated by these ribosomes. Consequently, the method requires no a priori knowledge of the translated sequences, enabling work with fully customizable transcript libraries rather than previously sequenced genomes. As a standardized framework for inverse toeprint generation, amplification and sequencing, iTP-seq can be used in combination with different types of libraries, in vitro translation conditions and data-analysis pipelines tailored to address a range of biological questions. Here, we present a robust protocol for iTP-seq and show how it can be integrated into a broader workflow to enable the study of context-dependent translation inhibitors, such as antibiotics. The time required to complete this workflow is ~10 d, and the workflow can be carried out by an experienced molecular biologist, with data analysis also requiring a working knowledge of command-line tools and Python scripts.

Optical tweezers use focused laser beams to manipulate small particles, primarily for force sensing. Recent advances in nanoscale-trapping approaches have enabled the development of multiplexed sensing applications, such as temperature and viscosity detection. Upconversion particles (UCPs) and, in particular, lanthanide-doped nano-/micro-crystals (~6 nm to 6 μm) exhibit particular anti-Stokes emission properties, which facilitate their visualization when trapped and the detection of changes to their properties based on temperature and orientation. Their ion resonance enhances the trapping force, enabling the manipulation of smaller particles and their use for force sensing. Here we provide step-by-step instructions to build UCP-based holographic optical tweezers systems, including super-resolved photonic force microscopy and fluorescence optical tweezers. We detail the characterization of the setup for subfemtonewton-scale force sensing and include nanoprobe functionalization, force sensitivity validation and comparison with known forces. We further include the procedures for temperature and viscosity sensing, such as calibrating polarized spectra, initiating UCP rotation and analyzing viscosity via spectral fluctuations. Applications, including nanoparticle-DNA-coated gold film interactions and temperature distribution near single cells, are shown as well. The procedure typically requires 6 days to complete and is suitable for users with expertise in photonics.

Nascent proteins begin to fold during their synthesis, while still attached to the ribosome. The dynamic nature of ribosome-nascent chain complexes (RNCs) poses a challenge for conventional structural biology approaches, limiting our understanding of dynamic cotranslational events. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful label-free technique for studying the conformational equilibria and refolding of full-length proteins with peptide resolution. However, the large size of the ribosome and the need for stable, highly homogeneous samples have hindered the application of HDX-MS to RNCs. Here we present a strategy for analysing conformational dynamics and interactors of Escherichia coli RNCs using HDX-MS. High-quality RNCs are obtained through the gentle lysis of high-density cultures expressing uniformly stalled ribosomes, followed by ultracentrifugation and tag-based affinity purification. Peptide-resolution information on protein conformational dynamics is obtained by pulse deuterium labeling, quenching with an RNA-compatible low pH buffer and offline digestion with pepsin. Extensive data analysis with use of specific internal controls allows for the confident assignment of mass spectra to specific peptides, ensuring good coverage of the nascent chain and ribosomal proteins. This method provides a valuable complement to existing structural techniques such as cryo-electron microscopy and nuclear magnetic resonance, and enables detailed characterization of large, partially structured nascent chains and their interactions with the ribosomal proteins and molecular chaperones. The protocol takes 1-3 months, from sample preparation and data acquisition to data analysis, and requires standard expertise in cloning and protein purification and intermediate expertise in HDX-MS.

Multimodal profiling of different molecular layers from the same single cell enables more comprehensive characterization of cellular heterogeneity compared with conventional single-modality approaches. A key example is co-detection of chromatin accessibility and gene expression that offers the opportunity to investigate cell type-resolved gene regulatory mechanisms. Here we describe a sensitive and robust protocol for in situ sequencing hetero RNA-DNA-hybrid after assay for transposase-accessible chromatin using sequencing (ISSAAC-seq) for the concurrent measurement of chromatin accessibility and gene expression from the same single nucleus. The method begins with dual Tn5 tagging of open chromatin regions and the RNA-cDNA hybrid produced by reverse transcription that take place in bulk nuclei. Then, various single-nucleus isolation strategies, including plate and droplet barcoding-based approaches, can be used based on the experimental purpose of the user. The protocol is highly modular with a flexible throughput ranging from several hundreds to tens of thousands of nuclei. The generated data are of high quality in both modalities. The entire workflow can be finished within 1 or 2 days, and the procedures work on multiple different single-nucleus isolation and barcoding platforms.

The isolation of small extracellular vesicles (sEVs), viruses and other nanoscale lipid particles from biofluids offers actionable possibilities for advancing disease diagnosis, drug delivery, regenerative medicine, personalized medicine and immunotherapy. Several methods are available to isolate sEVs from biofluids and acoustic techniques provide distinct advantages. Challenges constraining its wider application encompass the absence of adequate procedures for fabrication, implementation and performance validation. These issues impede the development of protocols applicable to nanoscale bioparticles experiencing acoustic isolation effects. Here we present a detailed protocol for acoustic separation of nanoscale bioparticles from biofluids, including plasma and saliva, achieving both high purity and throughput suitable for routine application. This protocol offers a comprehensive, step-by-step guide for the design and fabrication of the acoustic separation device, the establishment of the experimental setup and the isolation of bioparticles. To ensure reliability, rigor and reproducibility, we delineate essential procedures, including acoustic field optimization, channel fabrication and biofluid preparation, subsequently validating the protocol and its performance across different operators. Our protocol further encompasses procedures for data collection and analysis, which are essential for characterizing viruses and sEVs, as well as for evaluating their quality and integrity. This protocol enables researchers to perform high-quality isolation of nanoscale bioparticles, providing access to reliable acoustic separation techniques. Standardizing this technique will pave the way for discoveries in virology and intercellular communication research, with applications in medicine, biology, and materials science.