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Current Protocols is issuing a correction for the following protocol article.

Zhao, Y., & Hong, J. (2025). Expression, purification, and ATP hydrolysis activity measurement of CtISWI and CtATPase. Current Protocols, 5, e70241. doi: 10.1002/cpz1.70241

In the above-referenced article:

Figure 14 has been replaced with the corrected version.

The current version online now includes this correction and may be considered the authoritative version of record.

Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus

Support Protocol 1: Escherichia coli competent cell preparation and transformation

Support Protocol 2: A. rhizogenes competent cell preparation and transformation

Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus

Basic Protocol 3: Screening of transgenic hairy root lines

Support protocol 3: DNA isolation from L. sativus hairy roots

Chemotaxis is a fundamental biological process in which cells such as leukocytes migrate directionally from one site to another in response to a chemical gradient. This process of cellular migration is critical for execution of appropriate physiological responses to inflammation, as observed during immune defense and wound healing responses. Neutrophils being the first responders to the sites of infection or injury are one such type of cells. A neutrophil chemotaxis assay is a commonly used laboratory technique used to measure the directed movement of neutrophils toward a chemical signal termed a chemoattractant. It is used in immunology and inflammation research to study directed immune cell migration and to assess the effects of various molecules on cell migration. When studying neutrophil chemotaxis, current methods rely on counts of cells that have migrated across a membrane or substrate and observation of the migrated cells under a microscope. While useful, this approach fails to account for cells that have been activated but remain adherent to the substrate or cells that are in the intermediate stages of transmigration. Such limitations restrict the scope of information obtained and may lead to incomplete interpretations of neutrophil chemotaxis. To address this, we developed a new image analysis method that directly addresses the membrane in a chemotaxis chamber called the Boyden chamber. This improved technique allows quantification of neutrophils that have migrated as well as those that are adherent or actively transmigrating. By including all three stages of neutrophil movement, it provides a more comprehensive and physiologically relevant understanding of chemotaxis. It offers researchers a powerful tool to dissect the dynamics of immune cell behavior with greater sensitivity and accuracy, paving the way for deeper insights into immune responses and potential therapeutic interventions in inflammatory diseases. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Assay for the migration of bone marrow–derived neutrophils using the Boyden chamber

Basic Protocol 2: Analysis of adherent, transmigrating, and transmigrated cells

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