Current protocols最新文献

The chromatin immunoprecipitation followed by sequencing (ChIP-seq) assay is an instrumental and accurate method for understanding chromatin dynamics in eukaryotic cells. It provides critical insights into the regulation of gene expression and enables identification of regulatory elements, patterns of histone modifications, and chromatin states in health and disease conditions. Although cell cultures are great models to study molecular mechanisms associated with pathologies, studying tissues provides a physiologically native environment that reflects the cellular heterogeneity and spatial organization that are missing in an in vitro model. Several ChIP-seq protocols have been published; however, performing ChIP-seq in tissues remains a challenge in many settings due to the heterogeneity of tissues, complexity of cell matrices, low input material and intricacy of chromatin fragmentation and handling. Here, we present an optimized ChIP-seq protocol for solid tissues, with a focus on colorectal cancer. In this article, we incorporate simplified and efficient procedures for tissue preparation, chromatin extraction, immunoprecipitation, and library construction. The refined protocols overcome common limitations related to tissue processing and allows for highly reproducible, sensitive, and scalable analysis of disease-relevant chromatin states in vivo. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Frozen tissues preparation

Basic Protocol 2: Chromatin immunoprecipitation from tissues

Basic Protocol 3: Library construction for DNA sequencing

Basic Protocol 4: DNA nanoballs preparation for the DNBSEQ-G99RS sequencing platform and data quality control

Bulk RNA-sequencing (RNA-seq) is commonly used for identifying and characterizing genes through annotation, phylogeny, and differential expression analysis. For organisms without a reference genome, the need to generate a de novo transcriptome assembly for analyzing these data can prove challenging, as it is a multi-step process requiring many tools and computational resources. Therefore, a standardized and reproducible workflow using current best practices is required. We introduce the nf-core/denovotranscript workflow, an open-source solution built using Nextflow and the nf-core framework. The protocols here describe how the workflow can be used to perform pre-processing, de novo transcriptome assembly, redundancy reduction, assembly quality assessment, and quantification using RNA-seq data. This workflow offers simple installation and thorough documentation. The use of Docker, Singularity, and Podman container technologies also makes it portable across various computing environments. © 2025 Wiley Periodicals LLC.

Basic Protocol: Running nf-core/denovotranscript for de novo transcriptome assembly and quantification

Support Protocol: Installation and configuration for command line usage

This set of protocols was developed to detect Ascochyta rabiei, the fungus responsible for chickpea blight, in seeds. Given the significant impact of this disease and its potential to cause complete crop loss, even at low inoculum levels, optimizing detection protocols is crucial. This article proposes adjustments to key variables of a basic agar plate test—disinfection method, incubation, illumination, and culture medium—to improve microscopic identification (mainly using a stereomicroscope) and enhance differential growth of A. rabiei relative to other seed-borne fungi. Each step was carefully validated. Detection was verified through morphological and molecular confirmation of A. rabiei, as well as interlaboratory repeatability tests. In addition, the first distribution map of A. rabiei in Argentina was generated based on chickpea seed analyses conducted in the past decade and using the protocols outlined in this article. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Seed sampling procedure

Basic Protocol 2: Preparation of culture medium

Support Protocol: Sterilization of antibiotic

Basic Protocol 3: Seed disinfection by washing

Basic Protocol 4: Seed sowing and incubation

Basic Protocol 5: Detection of infected seeds

The cover image is based on the article Kinetic Analysis of HDAC- and Sirtuin-Mediated Deacylation on Chemically Defined Histones and Nucleosomes by Zhipeng Wang et al., https://doi.org/10.1002/cpz1.70251.

Histone deacetylases (HDACs) and sirtuins (SIRTs) play essential roles in regulating chromatin structure and gene expression by catalyzing the removal of acyl groups from histone lysine residues. Accurate characterization of their deacetylation kinetics is critical for understanding their enzymatic mechanisms and for guiding inhibitor or activator development. Given the complexity of enzyme-nucleosome core particle (NCP) interactions, including the influence of histone composition, post-translational modifications, and DNA context, NCP-based assays provide a more physiologically relevant platform than those performed on peptide or free histone substrates. Here, we present optimized protocols for assessing HDAC and SIRT deacetylation kinetics using NCP substrates, including determination of Michaelis-Menten parameters, evaluation of inhibitor and activator potency (IC₅₀ or EC₅₀), and instructions for ensuring assay reproducibility. These methods enable robust comparison of small-molecule modulators under conditions that better mimic the native chromatin environment, supporting both mechanistic studies and drug discovery efforts. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Assay of HDAC complex or SIRT deacylation on nucleosome substrates

Basic Protocol 2: Assay of HDAC complex or SIRT deacylation on free histone proteins

Basic Protocol 3: K_M measurement for deacylation assay on nucleosome or cofactor

Basic Protocol 4: Assessment of deacylation inhibitor or activator effects on NCP substrates

Genetic alphabet expansion by creating unnatural base pairs (UBPs) could renovate biological systems and next-generation biotechnologies. Among the expanded genetic letters, TPT3-NaM is one of the most advanced UBPs. It has been utilized in semi-synthetic organisms (SSOs) to recode therapeutic proteins. This article describes the synthesis of isoTAT and demonstrates that isoTAT can convert NaM to G, simultaneously pairing with both NaM and G as a bridging base. Additionally, the inherent base’ preference for NaM leads to its transformation into T. Consequently, TPT3-NaM can be converted to C-G or A-T base pairs through simple PCR assays, enabling the first method to locate multiple sites of TPT3-NaM pairs dually. Taken together, this work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs. The data presented in this article are based on our previously published reports. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of isoTAT triphosphate

Basic Protocol 2: Incorporation and extension reaction of isoTAT for a template containing NaM

Basic Protocol 3: Monitor DNA containing multiple UBPs using simple PCR assays with isoTAT through Sanger sequencing

Cardiac microtissues (cMTs) are three-dimensional, self-organizing structures that recapitulate key features of native heart tissue. cMTs can be generated using induced pluripotent stem cells (iPSCs) technology by combining iPSC-derived cardiomyocytes (CMs), endothelial cells (ECs), and cardiac fibroblasts (CFs) in a predefined ratio that closely resembles the cellular composition of the heart. Although cMTs lack true vascular perfusion, the incorporation of endothelial cells promotes the spontaneous formation of microvascular-like networks, which are identifiable within the 3D structure. The presence of vascular endothelial cells within cMTs enhances cardiomyocyte maturation and contraction force, while also making these models valuable in disease modeling, particularly for conditions involving endothelial dysfunction. High-resolution imaging of cMTs allows detailed visualization of cellular components and analysis of vascular-like structures, providing critical insights into the complex interactions between cardiac cells and their microenvironment. Here, we present protocols for the generation and examination of scaffold-free, vascularized cMTs derived from human iPSCs. First, we outline the steps for aggregation of pre-differentiated iPSC-CMs, iPSC-ECs, and iPSC-CFs to form vascularized cMTs. We then detail the downstream methodology for fluorescence labeling of both whole-mount and sectioned cMTs, followed by image acquisition, visualization, and capture using confocal microscopy. Finally, we demonstrate image analysis, and the measurement of the capillary-like networks using ImageJ (FIJI) software, with 3D modeling and Z-stack processing modules. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Formation of tri-lineage hiPSC-derived cardiac microtissues

Basic Protocol 2: Immunofluorescence staining of whole-mount and sectioned cardiac microtissues

Alternate Protocol: Counterstaining of whole-mount cardiac microtissues for cryo-sectioning

Basic Protocol 3: Cardiac microtissue imaging setup and data acquisition using confocal microscopy

Basic Protocol 4: Assessment of the vascular network in cardiac microtissues

Aptamer–drug conjugates (ApDCs) represent a powerful platform for targeted drug delivery, offering the potential to enhance therapeutic efficacy while minimizing systemic toxicity. However, conventional methods for ApDC preparation often involve complex, multistep procedures with limited control over drug loading and site specificity. This protocol describes a modular and automated strategy for the synthesis and structural optimization of ApDCs using custom-designed phosphoramidites that incorporate anticancer drugs and functional moieties. The customized phosphoramidite is compatible with standard solid-phase oligonucleotide synthesis, enabling the automated and efficient construction of programmable aptamer–drug conjugates (PApDCs) for targeted drug delivery. We also highlight the programmable optimization of aptamer structures to meet clinical needs. The protocol offers a streamlined and versatile platform for the construction of functional ApDCs, facilitating their application in receptor-targeted chemotherapy and nucleic acid–based therapeutics. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis and purification of phosphoramidites

Basic Protocol 2: Automated solid-phase synthesis and characterization of ApDCs

Unattended endotoxin (ETX) contamination in biological samples constitute a major challenge for in vitro and in vivo applications. Besides being potentially life-threatening, ETX contamination is especially relevant in global transcriptome analyses, where competing ETX stimulation can significantly skew the final gene expression profile. Our studies in mice and cultured skin epithelial cells (epidermal keratinocytes) aiming to characterize the effect of antibodies such as AK23 immunoglobulins (IgG directed against the cell-cell adhesion molecule desmoglein [DSG] 3) in the autoimmune disease pemphigus vulgaris (PV) revealed that laboratory-produced and even commercial control antibodies can exhibit non-negligible ETX contaminations. Moreover, these contaminants are extremely difficult to remove. To overcome these challenges, we have devised a simple yet nontoxic and scalable two-step protocol to efficiently reduce ETX levels during or after the IgG purification process. It consists firstly of 0.5 M NaOH pre-treatment of all devices, including the protein A resin, used during IgG sanitization and purification, in parallel with meticulous in-process monitoring of ETX levels. Secondly, before IgG elution from protein A, ETX is stripped from IgG by ion-exchange with the common amino acid arginine. This two-step approach successfully reduces ETX by >95% from hybridoma-derived, laboratory-produced AK23 IgG, as well as patient PV and control IgG, resulting in an 85% IgG recovery rate and ETX levels compatible with U.S. Pharmacopeia guidelines. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Sanitization of devices and protein A resin with 0.5 M NaOH

Basic Protocol 2: On-column stripping of ETX from AK23 IgG

Basic Protocol 3: Quality control of sanitized AK23 IgG

High-throughput screening (HTS) of proteins is used in a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimization, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high-throughput protein expression and screening methods (be that for expression, activity, stability, or binding assays), include the required yield, reproducibility, solubility, stability, purity, and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce enough protein of appropriate purity. This limits the type of assay and number of protein variants that can be simultaneously tested in an experiment. Here, we describe a HTS protocol that allows the overnight expression, export, and assay of recombinant proteins from Escherichia coli cells in the same microplate well. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture medium. The resulting protein is of sufficient purity and yield that it can be used directly in plate-based enzymatic assays without additional purification. This simple single-plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Expression, export, and isolation of vesicular-packaged recombinant protein

Support Protocol 1: 96-well plate cold-shock transformation

Support Protocol 2: In-plate affinity-tag protein purification

Support Protocol 3: Example in-plate enzymatic assay