Molecular Biology

In wheat and other crops, some genes display presence/absence variation, and it is occasionally beneficial to select for the absent allele to remove a functional gene. However, current high-throughput genotyping methods used to detect the absence of genes tend to be inconsistent and inconclusive. Kompetitive allele-specific PCR (KASP) and PCR allele competitive extension (PACE) are two well-established methods for allele-specific polymerase chain reaction (AS-PCR) assays, each using fluorescence resonance energy transfer (FRET) to generate a signal for each allele, typically targeting biallelic single-nucleotide polymorphisms. KASP and PACE methods are more difficult to apply to alleles with presence/absence variation because the lack of amplification of the absent allele is indistinguishable from a failed PCR. Here, we present a multiplex fluorescence-based absent allele–specific amplification (FAASA) method using the PACE marker system (compatible with KASP markers) to detect the absence of one particular or all alleles of a target sequence using a primer mix consisting of one target-specific primer pair (TSP) and a second primer set specific to a highly conserved endogenous gene known as a core gene–specific primer pair (CGSP). The forward primer of each pair is tagged with a 5′ terminal tail complementary to dye-labeled oligonucleotides in commercially available FRET cassettes. Lines that amplify only the core gene do not carry the target, while lines that amplify both the core gene and the target carry alleles of both the core gene and the target. The inclusion of the CGSPs allows researchers to confidently distinguish lines with absent alleles of the target from lines with failed PCR reactions, which can happen due to various reasons, including inadequate DNA quality or quantity.

Agrobacterium rhizogenes–mediated hairy root transformation provides a rapid platform for gene function analysis prior to stable whole-plant transformation. However, most existing hairy root transformation methods rely on tissue culture and require chemical or fluorescence-based selection, which increases experimental complexity. Here, we describe a tissue culture–free soybean hairy root transformation protocol incorporating the RUBY visual reporter system. While this work does not introduce a new transformation concept, it presents a streamlined implementation of established soybean hairy root methodologies that emphasizes procedural simplicity, reduced handling, and faster access to functional root material. Transgenic roots expressing RUBY can be directly identified by red pigmentation with the naked eye. In RUBY-positive roots, candidate genes driven by the CaMV 35S promoter showed higher expression levels than those in empty-vector controls, indicating that the system supports effective gene expression. Using this procedure, clearly identifiable transgenic hairy roots can be obtained within 20 days. Overall, this protocol simplifies induction and screening while reducing operational complexity and equipment requirements.

Extrachromosomal circular DNA (eccDNA) is a type of circular DNA that exists independently of chromosomes and has garnered significant attention in various fields, particularly in the context of smaller eccDNAs, which have considerable roles in gene regulation through various mechanisms. Current methods such as Circle-Seq and 3SEP can enrich small eccDNAs during sample preparation, but most bioinformatics pipelines remain challenging, exhibiting low accuracy and efficiency. This protocol describes the detailed workflow of a newly developed bioinformatics analysis pipeline, named EccDNA Caller based on Consecutive Full Pass (ECCFP), to accurately identify eccDNA from long-read Nanopore sequencing data. Compared to other pipelines, ECCFP significantly improves detection sensitivity, accuracy, and runtime efficiency. The process includes raw data quality control, trimming of adapters and barcodes, alignment to a reference genome, and identification of eccDNA, with detailed results encompassing accurate positioning of eccDNA, consensus sequences, and variants of individual eccDNA.

Transcription factors (TFs) regulate gene expression by binding to cis-regulatory elements in the genome. Understanding transcriptional regulation requires genome-wide characterization of TF occupancy across different chromatin contexts, yet simultaneous assessment of TF binding for multiple factors remains technically challenging. Here, we describe a detailed and reproducible protocol for cFOOT-seq, a cytosine deaminase–based genomic footprinting assay by sequencing, which enables antibody-independent, base-resolution profiling of chromatin accessibility, nucleosome organization, and TF occupancy. In cFOOT-seq, the double-stranded DNA (dsDNA) cytosine deaminase SsdA_tox converts cytosine to uracil in accessible chromatin, whereas TF binding and nucleosome occupancy locally protect DNA from deamination. Using the FootTrack analysis framework, deamination patterns generated by cFOOT-seq are quantitatively analyzed to derive standardized footprint and chromatin organization profiles at base resolution across the genome. Because cFOOT-seq preserves genomic DNA integrity during deamination-based footprinting, it is compatible with ATAC-seq-based chromatin enrichment. ATAC-combined implementations reduce sequencing depth requirements and improve scalability for footprint-focused analyses, supporting applications in low-input and single-cell settings. This protocol provides a practical framework for genome-wide TF footprint profiling and can be readily applied to dissect gene regulatory mechanisms in development, immunity, and disease, including cancer.

Amphibian retinas contain “green” rods, which are rod-shaped photoreceptors with a cone-type visual pigment. These rods are considered a potentially transitional photoreceptor type, but their phototransduction cascade’s molecular composition has remained uncertain. Here, we present a streamlined electrophysiology-molecular workflow that enables the rapid spectral identification, physical capture, and targeted single-cell reverse transcription-polymerase chain reaction (RT-PCR) of individual amphibian photoreceptors. After suction-pipette spectral screening under alternating red and green illumination, electrophysiologically identified cells are isolated and processed directly for reverse transcription and PCR. Coupling real-time functional phenotyping with sensitive molecular profiling provides a practical tool for resolving photoreceptor molecular heterogeneity and investigating evolutionary transitions between rod and cone phenotypes.

DNA epigenetic modifications play crucial roles in regulating gene expression and cellular function across diverse organisms. Among them, 5-glyceryl-methylcytosine (5gmC), a unique DNA modification first discovered in Chlamydomonas reinhardtii, represents a novel link between redox metabolism and epigenetic regulation. Accurate genome-wide detection of 5gmC is essential for investigating its biological functions, yet no streamlined method has been available. Here, we present deaminase-assisted sequencing (DEA-seq), a simple and robust approach for base-resolution mapping of 5gmC. DEA-seq employs a single DNA deaminase that efficiently converts unmodified cytosines (C) and 5-methylcytosine (5mC) into uracils or thymines, while leaving 5gmC intact. This selective resistance generates a clear sequence signature that enables precise identification of 5gmC sites across the genome. The method operates under mild reaction conditions and is compatible with low-input DNA, minimizing sample loss and improving detection sensitivity. Overall, DEA-seq provides an accessible, efficient, and highly accurate protocol for profiling 5gmC, offering clear advantages in workflow simplicity, DNA integrity, and analytical performance.

In the Japanese rhinoceros beetle Trypoxylus dichotomus, gene function studies have relied mainly on systemic larval RNA interference (RNAi), as gain-of-function techniques remain underdeveloped and germline transgenesis is impractical given the species’ approximately one-year generation time. In addition, because larval RNAi is systemic, it has been difficult to analyze the function of lethal genes. Here, we present a simple and efficient protocol for the direct introduction of exogenous DNA into T. dichotomus larvae via in vivo electroporation. This protocol includes optimized procedures for adult breeding and egg collection, as well as a rigorously parameterized electroporation technique that delivers a piggyBac transposon vector into region-specific larval tissues. Within one day after electroporation, treated larvae exhibit mosaic expression of a reporter gene, enabling rapid tissue-specific functional analysis without the need to establish stable germline transgenic lines. Moreover, the key promoter used in this system (T. dichotomus actinA3 promoter) is effective across diverse insect species, indicating that the method can be readily adapted to other non-model insects. Overall, this electroporation-based approach provides a valuable gain-of-function tool for T. dichotomus and potentially many other insect species.

Serial spatial omics technologies capture genome-wide gene expression patterns in thin tissue sections but lose spatial continuity along the third dimension. Reconstructing these two-dimensional measurements into coherent three-dimensional volumes is necessary to relate molecular domains, gradients, and tissue architecture within whole organs or embryos. sc3D is an open-source Python framework that registers consecutive spatial transcriptomic sections, interpolates bead coordinates in three dimensions, and stores the result in an AnnData object compatible with Scanpy. The workflow performs slice alignment, 3D reconstruction, optional downsampling, and interactive visualization in a napari-sc3D-viewer, enabling virtual in situ hybridization and spatial differential gene expression analysis. We tested sc3D on Slide-seq and Stereo-seq datasets, including E8.5 and E16.5 mouse embryos, recovering continuous tissue morphologies, cardiac anatomical markers, and the expected anterior–posterior gradients of Hox gene expression. These results show that sc3D allows reproducible reconstruction and analysis of volumetric spatial omics data across different samples and experimental platforms.

Our genome is duplicated during every round of cell division through the process of DNA replication, but this fundamental process is subjected to various stresses arising from endogenous or exogenous sources. Thus, studying replication dynamics is crucial for understanding the mechanisms underlying genome duplication in physiological and replication stress conditions. Earlier, radioisotope-based autoradiography and density-labeling methods were used to study replication dynamics, which were limited in spatial resolution, representing only average estimates from many DNA samples. Here, we describe a DNA fiber assay that utilizes different thymidine analog incorporation, like 5-chloro-2’-deoxyuridine (CldU) and 5-iodo-2’-deoxyuridine (IdU), into replicating DNA. Such labeled DNA can be stretched and fixed on silanized glass slides, which are denatured with mild acidic treatment to expose the labeled nascent DNA. This DNA can then be visualized by using primary antibodies against CldU and IdU, followed by fluorophore-conjugated secondary antibodies, and observing them using a fluorescence microscope. The DNA fiber assay allows the visualization of individually replicating DNA at a single-molecular resolution and is highly quantitative, high-throughput, and easily reproducible. This technique offers insights into different replication parameters, like rate of DNA synthesis, extent of reversed fork protection, restart of stalled forks, and fork asymmetry under untreated or replication stress conditions at a single-molecule level.

It is common practice for laboratories to discard clotted blood or freeze it for future DNA extraction after extracting serum from a serum-separating tube. If freezing for DNA extraction, the blood clot is not usually cryopreserved, which leads to cell membrane fragility. In this protocol, we describe steps to isolate high-quality nuclei from leukocytes derived from whole blood samples frozen without a cryoprotective medium. Nuclei isolated from this protocol were able to undergo ATAC (assay for transposase-accessible chromatin) sequencing to obtain chromatin accessibility data. We successfully characterized and isolated B cells and T cells from leukocytes isolated from previously frozen blood clot using Miltenyi’s gentleMACS Octo Dissociator coupled with flow sorting. Nuclei showed round, intact nuclear envelopes suitable for downstream applications, including bulk sequencing of nuclei or single-cell nuclei sequencing. We validated this protocol by performing bulk ATAC-seq.