Biology subjects

Sanchez, S.

Publications and source records attributed to Sanchez, S..

At least 19 recordsLinked to original sources

bioRxiv2026

3D-bioprinted skeletal muscle with tailored myofibre architecture

Engineered skeletal muscle tissue (eSMT) contractile function strongly depends on the alignment and orientation of its myofibres, as force generation and transmission are intrinsically linked to its microstructural architecture. However, fabricating truly functional eSMT with controlled myofibre architecture remains a major challenge. Here, we present a method that combines extrusion-based 3D bioprinting with custom multi-pillar anchoring systems to guide myofibre orientation. Using this approach, we successfully fabricated functional eSMTs with parallel, fusiform, bipennate, and circular architectures. We then integrated these basic designs into a functional system comprising multiple 3D-bioprinted eSMTs replicating the morphology and myofibre architecture of facial musculature. By recapitulating native muscle architecture, this approach advances the engineering of functional eSMT for multiple biomedical applications and establishes a foundation for next-generation biohybrid robotics.

bioengineering↗

bioRxiv2026

Combined lactate and hypothermia therapy enhances neuroprotection following neonatal hypoxic-ischemic brain injury

Background and Purpose: Neonatal hypoxia-ischemia (NHI) is a leading cause of mortality and long-term neurodevelopmental disability. Therapeutic hypothermia (TH) is currently the only established treatment for NHI; however, its efficacy remains limited, underscoring the need for adjunctive neuroprotective strategies. Lactate is now regarded as a major metabolic substrate of the brain and has previously been shown to exert neuroprotective effects following experimental NHI. We investigated whether lactate enhances hypothermia-induced neuroprotection and evaluated the optimal timing of administration and potential underlying mechanisms. Methods: Postnatal day 7 Wistar rats were subjected to NHI using the Rice-Vannucci model and treated with TH alone (2h at 32{degrees}C), sodium L-lactate alone, or combined L-lactate-TH. Animals underwent longitudinal magnetic resonance imaging (MRI) and behavioral assessments, complemented by histological, immunohistochemical, and Western blot analyses, including assessment of protein lactylation. The effect of sodium L-lactate administration timing relative to TH was also evaluated. Results: Lactate significantly enhanced the neuroprotective effects of TH, particularly when administered before hypothermia, resulting in greater lesion recovery, reduced neuroinflammation and apoptotic cell death, preserved neuronal integrity, and improved long-term motor and cognitive outcomes. Combined treatment preserved neuronal lactate transporter, prevented NHI-induced astrocytic glutamate transporter upregulation, modulated lactylation markers, supporting coordinated metabolic and epigenetic adaptations following NHI. Conclusions: Early lactate administration enhances the neuroprotective efficacy of TH following NHI. These findings identify lactate as a promising adjunct to TH and support its translational potential as a metabolic strategy to improve outcomes following NHI brain injury.

neuroscience↗

bioRxiv2026

Early Ontogenetic Development of Tessellated Calcified Cartilage in Chondrichthyans

Tessellated calcified cartilage (TCC) is a hallmark of the chondrichthyan skeleton, yet its development early in ontogeny across the four major groups (batoids, galeomorphs, squalomorphs, and holocephalans) remains poorly understood. Specialised traits of TCC, such as multi-layered TCC and internal mineralised trabeculae, typically develop in response to feeding mechanics. In this study, we evaluated TCC morphology in the jaws of 12 representative taxa to observe its structure at an early ontogenetic stage to determine whether these specialised features had yet developed. Batoids consistently exhibited well-developed, homogeneous, polygonal tesserae early in ontogeny regardless of jaw morphology or feeding habit. In contrast, galeomorphs displayed high morphological heterogeneity. Notably, we document the first report of an extensive internal trabecular network in a non-batoid elasmobranch, observed in Ginglymostoma cirratum, which may serve to resist the mechanical pressures of specialised suction feeding. Furthermore, we identified voussoir tesserae in galeomorphs for the first time, extending their documented presence across all elasmobranch groups, where they display an inverted aspect ratio (wider than tall) compared to mature forms. The durophagous Mustelus mustelus exhibited surprisingly poor TCC development despite being a durophagous feeder, pointing to a pronounced ontogenetic lag. In Squatina oculata, TCC was characterised by large and thick tesserae and extensive fused tesseral regions which may relate to its explosive ambush predation mode, whereas the holocephalan Chimaera exhibited a poorly mineralized, mesh-like structure without resolvable discrete tesserae or trabeculae-matching findings from previous studies. Across all specimens, multi-layered TCC was absent, confirming that multi-layering develops later in ontogeny. These results demonstrate that generalised models of TCC development based on one group or a few taxa fail to capture the broader diversity of TCC morphology. It also opens up many exciting avenues for further study, and forms the basis for comparisons with fossil chondrichthyans, to investigate the evolution of TCC.

zoology↗

bioRxiv2026

FAIRyMAGs - a series of FAIR Galaxy workflows for the generation of metagenome assembled genomes

Advances in whole-genome sequencing (WGS) technologies have enabled large-scale recovery of metagenome-assembled genomes (MAGs), providing unprecedented insights into microbial diversity across diverse environments. However, the reconstruction of MAGs remains computationally demanding and methodologically complex, requiring the integration of multiple tools for quality control, assembly, binning, refinement, and annotation. Existing workflows often rely on scripting-based implementations, constrain user-driven modification and stepwise execution, and require advanced expertise in high-performance computing (HPC) system administration, thereby limiting accessibility, reproducibility, and adaptability. Here, we present FAIRyMAGs, a Findable, Accessible, Interoperable, and Reusable (FAIR)-compliant, modular pipeline implemented within the Galaxy platform for the generation and analysis of MAGs. FAIRyMAGs consists of six interconnected workflows covering all major steps of MAG reconstruction, including read preprocessing, host and contaminant removal, assembly, binning, dereplication, and downstream taxonomic and functional annotation. The workflows are accompanied by extensive training material, including tutorials, a learning pathway, FAQs, test datasets and video walk-throughs by domain experts, supporting community adaptation. By leveraging Galaxys graphical interface and federated infrastructure, FAIRyMAGs enables users to execute complex analyses on public or private compute resources without requiring local installation or workflow programming expertise. The modular design further supports flexible adaptation, iterative optimization, and seamless integration of new tools contributed by the community. To demonstrate applicability, FAIRyMAGs was applied to four real-world microbiome datasets spanning various host-associated and environmental systems. These analyses revealed substantial variability in MAG recovery, community complexity, and clustering structure, underscoring the importance of flexible workflows adaptable to dataset-specific characteristics. Overall, FAIRyMAGs provides an accessible, extensible, and reproducible framework for genome-resolved metagenomics, reducing technical barriers and enabling methodological innovation through community-driven development within the adaptable Galaxy ecosystem.

bioinformatics↗

bioRxiv2026

Signaling input from the polar flagellum uncouples quorum sensing from cell-density regulation in Vibrio species

Quorum sensing (QS) is traditionally recognized as a signaling mechanism that monitors cell density to coordinate bacterial group behaviors. Although the architecture of many QS pathways and their dependence on cell density are well established, the impact from other physiological cues on QS response is largely unclear. We demonstrate here that flagellar integrity profoundly influences QS responses across multiple Vibrio species. Systematic disruption of flagellar components in Vibrio cholerae revealed that loss of the hook cap protein FlgD induces QS sRNAs Qrr1-4 production independent of cell density and the canonical QS regulator LuxO. This bypass mechanism also overrides normal cell density-dependent regulation of several QS-controlled phenotypes. Notably, {Delta}flgD mutation restores intestinal colonization of {Delta}luxO mutants in an infant mouse model, indicating that flagellar defects reshape V. cholerae QS during infection. This bypass mechanism is evolutionarily conserved as {Delta}flgD mutation induced LuxO-independent qrr expression in multiple Vibrio species. The FlrBC two-component system, essential for flagellar gene expression, is required for the activation of qrr expression in {Delta}flgD mutants. FlgD specifically interacts with the FlrB histidine kinase but not the FlrC response regulator, and a hyperactive FlrC variant is sufficient to drive qrr expression even in cells with FlgD. These findings suggest that not only FlrBC senses cytoplasmic FlgD levels to monitor flagellar completeness to direct flagellar gene expression, this signaling system also functions as a link to bypass the canonical cell-density control of QS by integrating cellular structural information to coordinate group behaviors.

microbiology↗

bioRxiv2026

Machine learning-based prediction of memory requirements for metagenomic assembly in high-performance computing environments

Metagenomic assembly can be a computationally intensive step in microbiome analysis, with memory requirements that vary widely depending on input data characteristics. In workflow systems like Galaxy and large-scale platforms like MGnify, which run thousands of heterogeneous jobs, inaccurate memory allocation drives job failures and costly retries when underestimated, and reduces throughput when overestimated. Current approaches rely primarily on heuristic rules based on input file size or sample metadata, which often fail to generalize across diverse datasets. In this study, we present a machine learning-based framework for predicting memory requirements of metagenomic assembly using metaSPAdes. We analyzed 300 assembly jobs from diverse biomes and evaluated 18 predictive models using combinations of input file size, biome classification, and sequence-derived k-mer features. K-mer profiles were computed from raw sequencing data and summarized into statistical descriptors capturing sequence complexity and diversity. Model performance was assessed using both conventional regression metrics and a production-oriented cost function that accounts for retry policies and resource waste in high-performance computing environments. Our results show that machine learning models can outperform commonly used heuristics. In particular, models incorporating biome information achieved the best performance and can be tuned to favor conservative predictions that reduce job failure rates. Simpler models based solely on input file size also performed competitively, offering a practical alternative for systems with limited feature availability. When evaluated under realistic workload distributions, predictive approaches reduced total memory waste by several million gigabyte-hours per 1,000 jobs compared to static allocation strategies. These findings demonstrate that data-driven resource prediction can substantially improve efficiency in metagenomic workflows. The proposed framework is adaptable to different computational environments and provides a foundation for integrating predictive resource allocation into large-scale bioinformatics platforms beyond Galaxy.

microbiology↗

bioRxiv2026

Decoding social integration in schooling fish using closed-loop real-virtual interactions

Collective motion in animal groups emerges from local interactions, yet how individuals select and integrate social information remains poorly understood. In schooling fish, inferring which neighbors drive an individuals behavior at any given moment is extremely challenging because the analysis of trajectories alone does not unambiguously reveal the underlying causal interactions that generate the observed motion. Here, we used a closed-loop virtual-reality system in which a single real rummy-nose tetra Hemigrammus rhodostomus interacted in real time with four model-driven virtual conspecifics whose interaction rules were precisely controlled. By systematically varying the number of influential neighbors of the virtual fish and combining experiments with simulations, we quantified how social information filtering shapes individual and collective dynamics. Collective coordination increased with stronger social coupling, but analyses of kinematics, spatial organization, correlations, and turning dynamics consistently showed that the real fish behaved as if it were responding primarily to the single most influential neighbor, whose identity could change over time. These results demonstrate that selective interaction with the most influential neighbor is sufficient to sustain coordinated group motion and highlight the power of bio-hybrid closed-loop experiments to reveal causal mechanisms of collective behavior.

animal behavior and cognition↗

bioRxiv2026

OKN4395, a first-in-class EP2/EP4/DP1 triple antagonist reprograms prostanoid-driven immunosuppression to restore antitumor immunity

Immune checkpoint inhibitors, particularly T cell targeting anti-PD(L)1 therapies, have revolutionized the treatment landscape for solid malignancies, but challenges related to non-responsiveness and the development of treatment resistance continue to be observed. An additional immunosuppressive axis relates to prostaglandin signaling downstream of cyclooxygenase-2 (COX2), where COX2 inhibitors have shown clinical promise in re-engaging both T and non-T cell immune compartments, yet have suffered from toxicity concerns. We report here the preclinical characterization of OKN4395, a highly potent and specific first-in-class triple antagonist of EP2, EP4, and DP1, major tumor immunosuppressive receptors downstream of COX2. OKN4395 restores immune function on both T cells and NK cells in vitro. Additionally, OKN4395 acts synergistically with anti-PD1 to increase speed and depth of antitumor activity. Overall, these findings robustly support the clinical investigation of OKN4395 in an ongoing Phase 1 trial (NCT06789172) as an innovative cancer immunotherapy for solid tumors, as a single agent and in combination with anti-PD1 therapy. Statement of significanceOKN4395, a first-in-class oral EP2/EP4/DP1 antagonist, reverses prostanoid-driven immunosuppression to restore antitumor immunity. Integrated pharmacology defines mechanism, translational biomarkers as well as both monotherapy and anti-PD1 combination strategies. These data position prostanoid tri-receptor antagonism as a translatable strategy in solid tumors. A global Phase 1 study is underway (NCT06789172).

pharmacology and toxicology↗

bioRxiv2026

Optimizing therapeutic hypothermia conditions in a translational preclinical model of neonatal hypoxia-ischemia in rats

BackgroundTherapeutic hypothermia is the only clinically approved treatment for neonatal hypoxia-ischemia (NHI), although its efficacy remains partial. In preclinical research, hypothermia is widely used as a reference therapy; however, its protocol is highly variable across studies, limiting robust comparisons with emerging neuroprotective strategies. This study aimed to define an optimal and standardized hypothermia protocol in the Rice-Vannucci model, not to challenge clinical practice, but to establish a reliable benchmark for preclinical therapeutic development. MethodsNHI was induced in postnatal day 7 (P7) rat pups, followed by normothermia or hypothermia for 2, 3, or 5 hours. Short- and long-term outcomes were assessed using lesion volume measurements by MRI, neurological scoring, behavioral tests, and histological analyses. The impact of immediate hypothermia initiation was also examined. ResultsAcross analyses, both 2- and 3-hour hypothermia durations provided greater neuroprotection than 5 hours--including brain lesion volume, motor and cognitive performances, and markers of neuronal preservation and neuroinflammation. However, for several parameters, 2 hours of hypothermia showed superior efficacy compared with 3 hours. Immediate initiation further modestly improved outcomes. ConclusionA 2-hour hypothermia protocol represents the most robust and reproducible preclinical reference, enabling meaningful comparison with novel therapies in the Rice-Vannucci model. IMPACTO_LIBy establishing an optimized hypothermia protocol in the Rice-Vannucci model, this study offers a consistent and robust reference for preclinical evaluation of emerging therapies. C_LIO_LIIt does not question clinical hypothermia protocols, but addresses variability in preclinical literature C_LIO_LIOptimizing the hypothermia reference protocol is mandatory to reliably identify new effective treatments in preclinical studies and to enhance their likelihood of successful and efficient clinical translation C_LI

neuroscience↗

bioRxiv2026

Swimming speed of schooling fish controls social interaction strength in open-loop immersive virtual reality

Collective motion in fish schools emerges from local attraction, alignment, and repulsion rules whose strengths vary with behavioural and biomechanical context. Although swimming speed is known to shape turning capacity, spatial positioning, and information flow, its causal influence on social interaction strength remains poorly quantified. Using an open-loop immersive virtual-reality system, we expose freely swimming Hemigrammus rhodostomus to a virtual conspecific moving along circular trajectories at six controlled speeds. This approach isolates the effect of neighbour speed on attraction, alignment, vertical positioning, and wall avoidance. Across all speeds, real fish reliably track the virtual fish and adjust their own speed toward that of the partner. Behavioural distributions reveal systematic changes in spatial positioning and perception angles, with fish shifting from leader-like to follower-like configurations as virtual-fish speed increases. A data-driven 3D self-propelled particle model, in which only the amplitudes of interaction functions are modulated, quantitatively reproduces these patterns. Model-based reconstructions shows that higher partner speed weakens both horizontal and vertical attraction, strengthens alignment, and increases wall repulsion. These results demonstrate that fish tune their social-interaction rules in a speed-dependent manner, revealing swimming speed as a key control parameter shaping coordination in minimal groups and, potentially, in larger schools.

animal behavior and cognition↗

bioRxiv2025

Closed-loop real-virtual interactions validate 3D model of social coordination in fish

Collective motion in animal groups arises from social interactions rules, yet uncovering these rules requires quantitative models grounded in real behavior. We developed a fully three-dimensional, data-driven model of pairwise interactions in the schooling fish Hemigrammus rhodostomus, reconstructing attraction, alignment from experiments with two real fish swimming freely in a hemispherical bowl. Simulations of this model quantitatively reproduced empirical distributions of speed, distance, and orientation. We then embedded the model into a closed-loop virtual reality system, allowing a real fish to interact in real time with a virtual conspecific whose movements were governed by the same interaction rules. This biohybrid setup revealed that the model captures key social interactions underlying coordinated swimming. Our results establish a robust validation framework linking data, models, and behavior, paving the way for hybrid biological-digital collectives.

animal behavior and cognition↗

bioRxiv2025

Dosing and Serostatus Shape the Efficacy of Adenovirus, mRNA, and Protein Vaccines

Despite the widespread use of adenovirus, mRNA, and protein-based vaccines during the COVID-19 pandemic, their relative immunological profiles and protective efficacies remain incompletely defined. Here, we compared antigen kinetics, innate and adaptive immune responses, and protective efficacy following Ad5, mRNA, and protein vaccination in mice. Ad5 induced the most sustained antigen expression, but mRNA induced the most potent interferon responses, associated with robust antigen presentation and costimulation. Unlike Ad5 vaccines, which were hindered by pre-existing vector immunity, mRNA vaccines retained efficacy after repeated use. As a single-dose regimen, Ad5 vaccines elicited superior immune responses. However, as a prime-boost regimen, and particularly in Ad5 seropositive mice, mRNA vaccines outperformed the other vaccine platforms. These findings highlight strengths of each vaccine platform and underscore the importance of host serostatus in determining optimal vaccine performance.

immunology↗

bioRxiv2025

Macropinocytosis inhibition attenuates pro-fibrotic responses in lung fibroblasts and pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in lung fibroblast activation and experimental pulmonary fibrosis. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from IPF patients. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) significantly inhibited profibrotic responses in IPF-derived and TGF-{beta}1-stimulated HLFs. EIPA exerted antifibrotic effects by regulating amino acid (AA) uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 (MEOX1) expression in activated HLFs. Both genetic and pharmacological inhibition of macropinocytosis significantly ameliorated pulmonary fibrosis in bleomycin (Bleo)-injured mice. Using IPF-derived precision cut lung slices (PCLS), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLS across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the Food and Drug Administration (FDA), effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice and IPF-derived PCLS. Taken together, our results suggest macropinocytosis inhibition as a potential therapeutic strategy to treat pulmonary fibrosis.

molecular biology↗

bioRxiv2025

A low-cost immersive Virtual Reality system to study social interactions and collective behavior in fish

1This study introduces a low-cost, open-source, immersive Virtual Reality (VR) system designed to investigate real-time social interactions and collective behaviors in fish. Understanding collective animal behaviors, such as schooling in fish, presents significant observational challenges due to rapid and complex interactions. To address these difficulties, we developed an innovative closed-loop VR environment allowing precise control and measurement of interactions between real and virtual fish. This setup incorporates high-speed 3D tracking, real-time visual feedback, and automated data processing, creating realistic and interactive scenarios. We present experimental results, obtained with rummy-nose tetras (Hemigrammus rhodostomus ), showing that freely moving real fish consistently adjusted their speed, depth, and spatial positioning to follow virtual fish effectively. Fish matched moderate virtual speeds comfortably, struggled slightly at higher speeds, and actively maintained vertical positioning to sustain group cohesion. The implementation of this VR system provides critical insights into sensory and cognitive processes underlying collective movement and decision-making in fish. By revealing both flexibility and constraints in fish behavior, our findings underscore the potential of VR technology to study social interactions in controlled yet realistic settings. Ultimately, this VR system advances our understanding of collective animal behavior, providing a foundation for cross-species studies linking neural processes, behavioral interactions, and group dynamics.

animal behavior and cognition↗

bioRxiv2024

Arabidopsis PRMT5 Buffers Pre-mRNA Splicing and Development Against Genetic Variation in Donor Splice Sites

Genetic variation at splice site signals significantly influences alternative splicing, leading to transcriptomic and proteomic diversity that enhances phenotypic plasticity and adaptation. However, novel splice variants can negatively impact gene expression and developmental stability. Canalization--the ability of an organism to maintain a consistent phenotype despite genetic or environmental variations--helps balance the effects of genetic variation on development and evolution. Protein arginine methyltransferase 5 (PRMT5) is a key splicing regulator in plants and animals. Most splicing changes in prmt5 mutants are linked to weak donor splice sites, suggesting that PRMT5 may buffer splicing against genetic variation. We examined PRMT5s effects on splicing and development in two genetically divergent Arabidopsis thaliana accessions with different single nucleotide polymorphisms (SNPs) affecting donor splice sites. While PRMT5 inactivation similarly affected splicing in both backgrounds, it significantly increased splicing and phenotypic differences between the accessions. Our findings suggest that PRMT5 contributes to canalization, mitigating the impact of splice site polymorphisms and facilitating the evolution of adaptive splicing patterns.

molecular biology↗

bioRxiv2024

Surface remodeling and inversion of cell-matrix interactions underlies community recognition and dispersal in Vibrio cholerae biofilms

Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. While it is commonly assumed that biofilm-dwelling cells are glued together by the matrix, how the cell-matrix interaction depends on the specific biochemistry of the matrix components and how this interaction varies during biofilm growth remains unclear. Here, we investigated cell-matrix interactions in Vibrio cholerae (Vc), the causative agent of cholera. We combine genetics, microscopy, simulation, and biochemical tools to show that Vc cells are not attractive to Vibrio polysaccharide (VPS), the main matrix component, but they can be bridged with each other and to the VPS network through crosslinking by Bap1. Downregulation of VPS and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results suggest a new conceptual model in understanding the intricate cell-matrix interaction as the major driver for biofilm development, which is potentially generalizable to certain other biofilm-forming species and exopolysaccharides.

biophysics↗

bioRxiv2024

Bioengineering Fascicle-like Skeletal Muscle Bioactuators via Pluronic-Assisted Co-axial 3D Bioprinting

Advances in 3D bioprinting have opened new possibilities in the development of bioengineered muscle models that mimic the structure and functionality of native tissues. The combination of skeletal muscle tissue and artificial elements promotes diverse innovative solutions of interest in both the biomedical field and the development of biohybrid actuators. However, current bioengineering approaches do not fully recreate the complex fascicle-like hierarchical organization of skeletal muscle, impacting on the muscle maturation process due to a lack of oxygen and nutrients supply in the scaffold inner regions. Here we explored co-axial 3D bioprinting as a strategy towards overcoming this challenge, creating individual/non-fused filaments with controlled thickness that present a fascicle-like organization. Compared to conventional 3D-bioprinting, where cell-laden bioink is disposed by a single syringe, our Pluronic-assisted co-axial 3D-bioprinting system (PACA-3D) creates a physical confinement of the bioink during the extrusion process, effectively obtaining thin and independent printed fibers with controlled shape. Fabrication of skeletal muscle-based actuators with PACA-3D resulted in improved cell differentiation, obtaining stronger bioactuators with increased force output when compared to bioactuators fabricated by conventional 3D bioprinting. The versatility of our technology has been demonstrated using different biomaterials, showing its potential to develop more complex biohybrid tissue-based architectures with improved functionality.

bioengineering↗

bioRxiv2024

Single-Molecule Barcoding Technology for Single-Cell Genomics

Recent advances in barcoding technologies have significantly enhanced the scalability of single-cell genomic experiments. However, large-scale experiments are still rare due to high costs, complex logistics, and laborintensive procedures. To facilitate the routine application of the largest scalability, it is critical to simplify the production and use of barcoding reagents. Here, we introduce AmpliDrop, a technology that initiates the barcoding process using a pool of inexpensive single-copy barcodes and integrates barcode multiplicity generation with tagging of cellular content into a single reaction driven by DNA polymerase during library preparation. The barcoding reactions are compartmentalized using an electronic pipette or a robotic or standalone liquid handling system. These innovations eliminate the need for barcoded beads and complex combinatorial indexing workflows and provide flexibility for a wide range of scales and tube formats, as well as compatibility with automation. We show that AmpliDrop is capable of capturing transcriptomes and chromatin accessibility, and it can also be adapted for user-customized applications, including antibody-based protein detection, bacterial or viral DNA detection, and CRISPR perturbations without dual guide RNA-expression vectors. We validated AmpliDrop by investigating the influence of short-term static culturing on cell composition in human forebrain organoids, revealing metabolic reprogramming in lineage progenitors.

molecular biology↗