Pinpointing causal genes for complex traits from genome-wide association studies (GWAS) remains a central challenge in crop genetics, particularly in species with extensive linkage disequilibrium (LD) such as rice. Here, we present CisTrans-ECAS, a computational protocol that overcomes this limitation by integrating population genomics and transcriptomics. The method’s core principle is the decomposition of gene expression into two distinct components: a cis-expression component (cis-EC), regulated by local genetic variants, and a trans-expression component (trans-EC), influenced by distal genetic factors. By testing the association of both components with a phenotype, CisTrans-ECAS establishes a dual-evidence framework that substantially improves the reliability of causal inference. This protocol details the complete workflow, demonstrating its power not only to identify causal genes at loci with weak GWAS signals but also to systematically reconstruct gene regulatory networks. It provides a robust and powerful tool for advancing crop functional genomics and molecular breeding.Key features• Pinpointing causal genes with high precision: Integrates cis- and trans-expression components to distinguish true causal genes from LD artifacts, even for small-effect loci.• Reconstructing gene regulatory networks: Uses gene expression as molecular traits to identify upstream regulators, revealing complex molecular regulatory pathways.• Versatile and reproducible workflow: An R-based pipeline using PLINK and GCTA, applicable to rice and other species with population genomics and transcriptomics data.• Experimentally validated reliability: The method successfully identified key genes OsMADS17 and SDT that regulate rice spikelet number, with their regulatory relationship confirmed by molecular experiments.

Nanoparticle vaccines can provide advantages over traditional vaccine methodologies, including adjuvant delivery to enhance the effectiveness of recombinant antigens. Many approaches exist to formulate different vaccine nanoparticles, which are designed for different biomolecular cargos, adjuvant compositions, and disease targets. Here, a protocol is described to produce nanoliposomes whose surface is decorated with recombinant protein influenza antigens with monophosphoryl lipid A and QS-21 adjuvants incorporated into the lipid bilayer for protection against influenza infection. This protocol includes methods for producing adjuvanted liposomes and coupling with His-tagged antigens for surface decoration of the particle. This allows for a rapid methodology of producing immunogenic antigen-presenting liposomes that can be tailored to display a combination of influenza surface antigens. Key features • This protocol uses recombinant proteins, which can be adapted to the desired strain sequence. • Production of this influenza vaccine formulation uses in vitro techniques, eliminating the need for virus growth in eggs. • Additional lipid adjuvants can be included in the liposome production step to be incorporated into the surface membrane. • Cobalt-porphyrin phospholipid provides a flexible platform for binding His-tag-bearing proteins.

The deletion and mutation of Topoisomerase 3β (TOP3B) is linked to multiple neurological disorders and is the only known topoisomerase that is also catalytically active on RNA in vitro and in cells. Uniquely, TOP3B is primarily localized to the cytoplasm, binds to open reading frames of mRNA, and regulates mRNA stability and translation in a transcript-specific manner. A common approach for studying TOP3B activity in cells is immunodetection of TOP3B•RNA covalent intermediates after bulk RNA isolation. However, in this approach, the RNA species is unknown and is not selective for the major TOP3B substrate, mRNA. In this protocol, we describe a recently developed and optimized protocol for capturing TOP3B•mRNA covalent intermediates using oligo-dT isolation of mRNA under protein-denaturing conditions. Covalent intermediates are then detected by a dual membrane slot blotting strategy with nitrocellulose and positively charged nylon membranes. Nitrocellulose membrane-bound TOP3B•mRNA covalent intermediates are analyzed by immunodetection, and nylon membrane-bound free mRNA is stained with methylene blue. The protocol detailed below has been validated with wildtype and mutant 3xFLAG-tagged TOP3B expressed in Neuro2A cells, with additional optimization for slot blotting using recombinant EGFP.Key features• This protocol is optimized for isolation of TOP3B•mRNA covalent intermediates from cultured mammalian cells.• Slot blotting allows for higher throughput and sensitive detection of TOP3B•mRNA covalent intermediates and allows for free mRNA to serve as a loading control.• Alternative to laborious extraction methods that do not select for TOP3B covalently linked to mRNA over other RNA species.• Can be completed in two days (not including variable time for mammalian cell sample collection).

ER-phagy, a selective autophagy process crucial for maintaining cellular homeostasis by targeting the endoplasmic reticulum (ER), has been challenging to study in vivo due to the lack of suitable spatiotemporal quantification tools. Existing methods like electron microscopy, biochemical assays, and in vitro reporters lack resolution, scalability, or physiological relevance. Here, we present a detailed protocol for generating two transgenic mouse models: ER-TRG (constitutively expressing an ER lumen-targeting tandem RFP-GFP tag) and CA-ER-TRG (Cre-recombinase-activated ER-TRG). Additionally, we outline procedures for quantitative imaging of ER-phagy in vivo, covering tissue preparation, confocal microscopy, and signal analysis. This protocol offers a robust and reproducible tool for investigating ER-phagy dynamics across various tissues, developmental stages, and pathophysiological conditions, facilitating both fundamental and translational research.Key features• Enables live, single-cell resolution imaging of ER-phagy dynamics across intact tissues in mice.• Features a Cre-recombinase-activated knock-in model (CA-ER-TRG) for spatiotemporally controlled ER-phagy studies in specific cell types.• Quantifies ER-phagy flux via pH-sensitive RFP-GFP signal ratiometry and lysosomal co-localization in vivo.

BrU-labeling and immunoprecipitation of newly synthesized RNA Anne Kruse Hollensen1, Christian Kroun Damgaard11Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark ProtocolSeeding of cellsSeed cells in eight p6 dishes per series of samples6 ml cell culture medium/dish PulseAspirate 4 ml of medium from each plate and pool medium from each series of samples. For each series of samples:For one dish (–BrU): Aspirate the remaining medium from the cells and add 3 ml of the collected medium.For the remaining four dishes (+BrU): Add 0.25 M 5-bromouridine (BrU) to a final concentration of 2 mMAspirate the remaining medium from the cells Add 3 ml +BrU medium to each plate Incubate for 1 hour ChaseAspirate medium Add 4 ml medium to the cells and aspirate Add 4 ml medium to the cells Incubate at 37˚ C for 5 min Aspirate Add 4 ml medium Incubate at 37˚ C Harvest the –BrU samples after 35 minutes Harvest the 0 h +BrU samples after 50 minutes Harvest the remaining samples after additionally 3 h, 6 h, and 9 h Harvest cellsPlace cell-plates on ice Carefully wash each plate once in cold PBS Lyse the cells in 1 ml cold Trizol Transfer the lysate to an Eppendorf tube Store at -20˚ C until purification of RNA RNA purificationPurify RNA according to Trizol protocol Resuspend RNA in nuclease free H2O Measure concentrations Dilute to 1 µg/µl   Immunoprecipitation of BrU-labeled RNAReagents:Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen)1xBrU-IP Buffer w. 1 mg/ml Heparin (see recipe below)Heparin (1 mg/ml)a-BrdU antibody, clone 3D4 (555627, BD Pharmingen)1xBrU-IP Buffer (see recipe below)BSA (100 mg/ml)0.25 M 5-bromouridine (5-BrU)BrU-labeled total RNA at 1 mg/mlRiboLock (40 U/µl)Phenol:Chloroform pH 6.6Chloroform3M NaAcGlycogen (10 µg/µl) Equipment:RotatorMagnetic standTable-top centrifugeThermo-shaker at 80˚ CCold centrifugeDry iceIce trayBuffers:Use RNase-free H2O for all buffers 2xBrU-IP buffer: 40 mM Tris-HCl pH 7.5500 mM NaCl 2xBrU-IP buffer + BSA/RiboLock:2x BrU-IP buffer1 µg/µl BSA80 U/ml RiboLock 1xBrU-IP buffer + Heparin:1xBrU-IP buffer1 mg/ml Heparin 1xBrU-IP buffer:1xBrU-IP buffer0.5 µg/µl BSA20 U/ml RiboLockElution buffer:0.1 % SDS Prepare beads/antibody:Beads/antibody are prepared in batch followed by aliquoting (beads: 20 µl/sample, antibody: 2.5 µl/sample)Resuspend the Goat anti-mouse IgG dynabeads thoroughly in the vial to obtain a uniform brown suspensionTransfer the desired volume of Dynabeads to an Eppendorf tube Spin briefly in table-centrifugePlace the tube on a magnet for 1-2 min Remove supernatant Remove the tube from the magnetWash beads (add 400 µL 1xBrU-IP buffer  mix by pipetting carefully a couple of times  rotate 2 minutes at room temperature  spin briefly in table-centrifuge  place the tube on a magnet for 1-2 min  remove supernatant) Repeat the wash Resuspend the beads in 1 ml 1xBrU-IP buffer with Heparin Rotate 30 min at room temperatureWash beads in 1xBrU-IP bufferResuspend the beads in 1 ml 1xBrU-IP bufferMix by pipetting carefully a couple of timesAdd the desired volume of a-BrdU antibody to the mixtureWash beads three times in 1xBrU-IP buffer Resuspend beads in 50 µl 1xBrU-IP buffer/sample Add 0.25 M 5-BrU to a final concentration of 1 mM BrURotate 30 minutes at room temperatureWash beads three times in 1xBrU-IP bufferResuspend beads in 50 µl 1xBrU-IP buffer/sample Prepare RNA samples:Transfer 40 µg (1 µg/µl) RNA for each sample to new Eppendorf tubes Add 160 µl H2ODenature the RNA by incubating at 80˚ C for 2 min Spin down briefly in table-centrifuge Add 200 µl 2xBrU-IP buffer with BSA/RiboLock Bind RNA:Add 50 µl resuspended prepared beads to each RNA sample Rotate 1 h at room temperature Spin down briefly in table-centrifugePlace the tube on a magnet for 1-2 min Carefully remove supernatant and transfer to Eppendorf tubes (unbound RNA, store at -20˚ C)Wash beads four times in 1xBrU-IP bufferResuspend the beads in 200 µl Elution buffer and proceed quickly to “Elute bound RNA” Elute bound RNA:Add 200 µl phenol/chloroform pH 6.6 to the resuspended beads Vortex Spin for 3 min at full speed at 4˚ C Transfer the supernatant (190 µl) to a tube containing 200 µl chloroformVortex Spin for 3 min at full speed at 4˚ C Transfer supernatant (180 µl) to a tube containing 18 µl 3M NaAc pH 6.0 and 2 µl glycogen (10 µg/µl) Add 500 µl 96% EtOH Mix by inverting the tube a couple of times Place on dry ice for 15 min or at -20˚ C for >1 hour Spin at full speed at 4˚ C for >30 min Discard supernatant completely without disturbing the pellet Wash with 185 µl 75% EtOH Spin at full speed at 4˚ C for 5 min Discard supernatant completely without disturbing the pellet Dry the RNA pellet Resuspend the RNA in 10 µl nuclease free H2O ReferencesHollensen, A.K., Thomsen, H.S., Lloret-Llinares, M., Kamstrup, A.B., Jensen, J.M., Luckmann, M., Birkmose, N., Palmfeldt, J., Jensen, T.H., Hansen, T.B., and Damgaard, C.K. (2020). circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation. Elife 9. 10.7554/eLife.58478. Kofoed, R.H., Betzer, C., Lykke-Andersen, S., Molska, E., and Jensen, P.H. (2018). Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation. J Vis Exp. 10.3791/57056.

The compound eyes of Drosophila are widely used to gain valuable insights into genetics, developmental biology, cell biology, disease biology, and gene regulation. Various parameters, such as eye size, pigmentation loss, formation of necrotic patches, and disorientation, fusion, or disruption of ommatidial arrays, are commonly assessed to evaluate eye development and degeneration. We developed an improved imaging method named low-angle ring illumination stereomicroscopy (LARIS) to capture high-contrast images of the Drosophila compound eye. Different optical alignments were tested to capture the fly compound eye image under the stereomicroscope; the highest contrast with minimal reflection was achieved through the LARIS method. The images captured using LARIS clearly showed ommatidial fusion, disorientation, and pigmentation loss, which were hardly visible with a conventional imaging method in the degenerating compound eyes of Drosophila. In addition to its research applications, this protocol is cost-effective due to the low expenses associated with supplies and equipment. We anticipate that LARIS will facilitate high-contrast imaging of the compound eyes in Drosophila and other insects.Key features• Low-angle ring illumination stereomicroscopy (LARIS) is an improved optical alignment for high-contrast imaging of Drosophila compound eyes.• LARIS is a simple, inexpensive, and robust method with additional advantages over existing SEM methods to capture high-contrast microscopic images of the Drosophila eye.• LARIS can be easily implemented across laboratories and used as a low-cost teaching/research tool.

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.Key features• A robust, affordable, high-throughput genotyping method for genes or other target sequences with presence/absence variation.• FAASA markers can be easily incorporated into established marker-assisted selection programs in labs using KASP and/or PACE markers.• FAASA markers can also be used for other genotyping applications like GWAS, QTL, or bi-parental mapping studies.• Easily adaptable to different targets and species of interest.

The morphology of single-neuron axonal projections is critical for deciphering neural circuitry and information flow in the brain. Yet, manually reconstructing these complex, long-range projections from high-throughput whole-brain imaging data remains an exceptionally labor-intensive and time-consuming task. Here, we developed a points assignment-based method for axonal reconstruction, named PointTree. PointTree enables the precise identification of the individual axons from densely packed axonal population using a minimal information flow tree model to suppress the snowball effect of reconstruction errors. In this protocol, we have elaborated on how to configure the required environment for PointTree software, prepare suitable data for it, and run the software. This protocol can assist neuroscience researchers in more easily and rapidly obtaining the reconstruction results of neuronal axons.Key features• Optimized for mapping long-range axons that connect distant brain regions in dense or crossover scenarios.• Enables high-fidelity (F1-score > 80%) reconstruction of hundreds of GB of large-volume imaging data.• Compatible with LSM, fMOST, and HD-fMOST systems for diverse neuroimaging datasets.