Nature Protocols最新文献

BreakTag is a scalable next-generation sequencing-based method for the unbiased characterization of programmable nucleases and guide RNAs at multiple levels. BreakTag allows off-target nomination, nuclease activity assessment and the characterization of scission profile, that, in Cas9-based gene editing, is mechanistically linked with the indel repair outcome. The method relies on digestion of genomic DNA by Cas9 and guide RNAs in ribonucleoprotein format, followed by enrichment of blunt and staggered DNA double-strand breaks generated by CRISPR nucleases at on- and off-target sequences. Next-generation sequencing and data analysis with BreakInspectoR allows high-throughput characterization of Cas nuclease activity, specificity, protospacer adjacent motif frequency and scission profile. Here we first describe a detailed BreakTag protocol for the nomination of CRISPR off-targets and multilevel characterization of engineered Cas variants and second, we describe a step-by-step protocol for data analysis using BreakInspectoR. Third, we provide a web interface for XGScission, a machine learning model amenable to training with scission-aware BreakTag data to predict the relative frequency of blunt and staggered double-strand breaks at new sequences unseen by the model. XGScission allows a preselection of target sequences predicted to be cut in staggered configuration that are preferably repaired as single-nucleotide templated insertions. Furthermore, XGScisson can be used to assess sequence determinants of blunt and staggered cleavage by SpCas9 and engineered nuclease variants. As a companion strategy, we describe HiPlex for the generation of hundreds to thousands of single guide RNAs in pooled format for the production of robust BreakTag datasets. The BreakTag library preparation takes ~6 h, and the entire protocol can be completed in ~3 d, including sequencing, data analysis with BreakInspectoR and XGScission model training.

This protocol provides comprehensive guidance for researchers, including those with limited electron microscopy (EM) experience, on the effective use of genetically encoded, multichannel EMcapsulin reporters in both fluorescence microscopy and EM workflows. EMcapsulins are a set of modular gene reporters that manifest as distinct shapes in EM when expressed in cell culture or model organisms. This protocol encompasses detailed instructions for labeling cells or proteins of interest with fluorescent EMcapsulins, along with biochemical quality control measures. Researchers may also adapt the protocol to develop custom EMcapsulin constructs tailored to their specific experimental requirements.

Radiative cooling is a renewable cooling mechanism with diverse applications. Many radiative cooling materials have been developed (e.g., fabrics, paints and building materials). It is, however, difficult to compare their properties because of a lack of standardized and transparent performance-evaluation systems. Here, we present procedures for assessing the optical and thermal properties of radiative cooling materials that can be applied to various forms such as films, coatings and fabrics. In the first procedure, hemispherical reflectance and transmittance spectra are collected by using two integrating sphere spectrometers for the solar spectrum (0.3-2.5 μm) and the IR spectrum (2.5-20 μm), respectively (2 h). We then describe how to build an outdoor performance-testing platform designed to accurately measure differences in temperature corresponding to the presence of different materials over extended periods. Thermal insulation and radiation shielding are fundamental requirements for this platform. Other environmental conditions are measured (e.g., humidity, sunlight, wind and external temperature). We also describe how to construct and use a smaller, indoor testing platform. Although it is challenging to fully replicate outdoor conditions, indoor testing still provides a standardized reference. Both platforms can be seamlessly integrated with a Proportional-Integral-Derivative temperature control system, enabling the simulation of applications in intricate thermal environments. Outdoor and indoor experiments typically take 7 and 1 d, respectively. Finally, we provide a simple MATLAB-based code for rapid theoretical performance evaluation (~10 min).

There is increasing interest in measuring the effect of microplastics and nanoplastics (MNPs) in the environment, but it is difficult to compare the results obtained in these studies due to variations in the extraction and characterization techniques, as well as the variability of the matrices analyzed. Here we provide a workflow consisting of three separate procedures for (1) preprocessing of different environmental samples, (2) methods for MNP extraction (four-step extraction method) and (3) techniques for qualitative and quantitative characterization of MNPs. The four-step extraction method (FSEM) involves predigestion, predensity separation, postdigestion and postdensity separation. This process has been optimized to maximize recovery (between 83.7% and 100% for polyethylene, polyethylene terephthalate, polypropylene, polystyrene and polymethyl methacrylate) and purity while minimizing artefactual changes to the particles. It is crucial to characterize the MNPs extracted using the FSEM to understand their chemical composition and other physicochemical properties such as quantity, particle size and morphology. We provide guidance on the use of different fit-for-purpose analytical technologies, including attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), laser direct infrared spectroscopy (LDIR) and optical photothermal infrared microspectroscopy (O-PTIR). These techniques can be combined to characterize MNPs with particle sizes of 0.5-5,000 µm. We provide advice on how to optimize the sample preparation methods by adding or removing extraction steps based on the complexity of the matrix and purpose of the analysis. The suggested validation workflow also uses additional analytical techniques (for example, atomic force microscopy and flow cytometry) to evaluate efficiency, feasibility and reliability. We provide experimental detail for analysis using micro-FTIR, which can be used instead of LDIR and O-PTIR to characterize microplastics with particle sizes larger than 10 µm. Execution of this workflow takes 7-30 d and can be performed by researchers, technicians and students in the environmental field if they have access to the required equipment.

Proteolysis-targeting chimeras (PROTACs) are traditionally conceptualized and synthesized by connecting two ligands, one for a target protein and one for an E3 ligase, via a bifunctional linker. Chemical creativity has recently explored the development of more elaborate and unusual linkers beyond conventional bifunctionals, allowing the development of macrocyclic and trivalent PROTACs. In two distinct proof-of-concept studies guided by the co-crystal structure of bivalent PROTAC MZ1 in complex with the E3 ubiquitin ligase von Hippel-Lindau and the second bromodomain of Brd4, we designed macrocyclic macroPROTAC-1 and trivalent PROTAC SIM1. These designs aimed to enhance protein degradation by constraining the PROTAC in its bioactive conformation or increasing avidity and cooperativity within the PROTAC ternary complex by augmenting the binding valency to the target protein, respectively. Here we describe the step-by-step synthesis of the macrocyclic macroPROTAC-1 and trivalent PROTAC SIM1, detailing the generation of the macrocyclic and trivalent cores and their subsequent conjugation to the respective ligands. This two-part procedure is expected to take ~14 d for the synthesis of macroPROTAC-1 and 10 d for the synthesis of SIM1. In this protocol, we also provide a brief introduction into the biophysical and cellular evaluation of these unusual molecules, representative structures of key negative control compounds and their utility, and highlight recent developments and expansion beyond pioneering exemplars.

Human pluripotent stem (hPS) cell-derived blood-generating heart-forming organoids (BG-HFOs) represent a highly structured in vitro model that recapitulates aspects of human cardiac, hematoendothelial and multiwave hematopoietic co-development. Our model offers novel insights into human development and represents a potent tool for disease modeling, drug testing and other advanced in vitro assays. Here we provide a detailed 14-d protocol for the generation of BG-HFOs, from the embedding of hPS cell-derived aggregates in Matrigel, directing cell differentiation via WNT pathway modulation and supplementation of cytokine cocktails required for hematoendothelial induction and maturation. The protocol is robust and applicable to different hPS cell lines. Proper formation and patterning of the multiple tissues present in the BG-HFOs can be assessed through techniques such as live-cell imaging, whole-mount immunofluorescence (IF) staining, flow cytometry and gene-expression analysis. The efficient generation of BG-HFOs requires hands-on experience with hPS cell culture and the simultaneous management of multiple medium-enriching growth factors and small molecules. We additionally highlight a simple, scalable method of sample preparation for the effective investigation of BG-HFO morphology via laser microscopy using common laboratory equipment. This Protocol requires basic skills in handling hazardous chemicals and limited experience in IF staining and laser microscopy. Our robust and fast method enables the whole-mount IF staining and clearing of large organoids (up to 4 mm in diameter) composed of multiple tissues (with different physical properties) such as BG-HFOs and other complex organoid models in 2.5 d. The described workflow can thus promote fast laser microscopy, thereby tackling a major challenge in the field.

Slippery covalently attached liquid surfaces (SCALS, also called quasi-liquid surfaces, liquid-like surfaces and slippery omniphobic covalently attached surfaces) have recently emerged as a new family of materials with many useful properties such as droplet-shedding, deicing and antifouling. They can also serve as model systems in studies of wetting, evaporation and self-assembly phenomena. They are made of nano-thin layers of polymers or oligomers that are liquid at ambient temperature and covalently attached to a smooth substrate. Here we lay out procedures for preparation of the most common SCALS system: polydimethylsiloxane bound to silica surfaces via silane chemistry. The apparent simplicity of these layers and their methods of preparation is misleading, and obtaining reproducible results requires careful control of the reaction parameters. The exact details of the synthetic methods for SCALS determine the observed results; it is therefore essential to report both the synthetic detail and the characterization results to improve reproducibility and to advance understanding of the field. Here a range of synthetic methods used in the literature were reproduced in three different laboratories across the world, their comparative advantages and disadvantages discussed and the resulting SCALS characterized. For each synthetic method, the key parameters that contribute to their performance and ease of reproducibility were identified and optimized.

Cytometry-based single-cell proteomics (SCP) has emerged as a powerful technique that greatly advances our understanding of complex biological systems with a new level of granularity. Various methods have been developed to process cytometry-based SCP data. However, it remains extremely challenging to identify the well-performing processing workflows for specific datasets. Here, we develop ANPELA, an out-of-the-box method for navigating the proteomic data processing based on large-scale screening. It enables a comparison among the performances of thousands of the processing workflows in identifying cell subpopulations and inferring pseudo-time trajectories based on machine learning. Several cases are then analyzed, highlighting its ability to identify the optimal ways of data processing for cytometry-based SCP studies. A new package is also deployed to ensure multiscenario usability (such as desktop software, R package and online server), data security (enabling local and open-source execution) and a user-friendly interface (realizing interactive and visualizable applications). Overall, ANPELA can be utilized by a broad audience, including those without coding skills, and is freely accessible and downloadable at https://idrblab.org/anpela/ . Its execution time may range from minutes to hours depending on the size of the analyzed data.

Volumetric printing is an emerging additive manufacturing technique that builds 3D constructs with enhanced printing speed and surface quality by forgoing the stepwise ink renewal. Existing volumetric printing techniques almost exclusively rely on light energy to trigger photopolymerization in transparent inks, limiting the material choice, build size, cell density and in vivo printability. Sonicated ink (or sono-ink) and focused-ultrasound (FUS) writing have been developed for deep-penetration acoustic volumetric printing (DAVP) within optically scattering media and beneath soft tissues. This technology uses rapid sono-thermal heating to induce material solidification at the FUS focal region, constructing 3D objects without the need for a build platform. Here, we describe two procedures necessary to achieve DAVP. First, we provide a step-by-step guide for preparing and characterizing multicomponent viscoelastic self-enhancing sono-inks. The lower critical solution temperature polymers are synthesized as a phase-transition reversible acoustic absorber to formulate the sono-inks. We characterize the rheological, acoustic and cytocompatibility properties of the sono-inks. We then detail the procedure for building a 3D FUS printer by integrating an FUS transducer with a 3D printing platform. The development of the 3D FUS printer needs basic knowledge of the ultrasound system, FUS physics and volumetric printing. Using the sono-inks and the 3D FUS printer, we further provide guidance to evaluate the sono-thermal heating effect and characterize the volumetric printing resolutions. We demonstrate the printing of volumetric constructs through optically scattering materials such as centimeter-thick biological tissues. The procedures require ~470 h to complete.