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Optimized Analytical Workflow for Single-Nucleus Transcriptomics in Main Metabolic Tissues

Single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful approach for studying cellular heterogeneity in metabolic tissues. However, snRNA-seq analysis remains challenging due to low gene expression and data complexity. Here, we introduce an optimized analytical workflow for snRNA-seq data from 67 samples across four main metabolic tissues white adipose tissue, hypothalamus, muscle and liver. We emphasized the importance of key steps including ambient RNA removal, doublet identification, normalization and data integration to ensure accurate downstream analysis. This workflow offers a valuable resource for researchers in metabolism, facilitating deeper insights into cellular diversity and metabolic function through rigorous snRNA-seq analysis.

Source Data: Visualization of chromosomal reorganization induced by heterologous fusions in the mammalian nucleus

Source data from Visualization of chromosomal reorganization induced by heterologous fusions in the mammalian nucleus

Optimized Analytical Workflow for Single-Nucleus Transcriptomics in Main Metabolic Tissues

Single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful approach for studying cellular heterogeneity in metabolic tissues. However, snRNA-seq analysis remains challenging due to low gene expression and data complexity. Here, we introduce an optimized analytical workflow for snRNA-seq data from 67 samples across four main metabolic tissues white adipose tissue, hypothalamus, muscle and liver. We emphasized the importance of key steps including ambient RNA removal, doublet identification, normalization and data integration to ensure accurate downstream analysis. This workflow offers a valuable resource for researchers in metabolism, facilitating deeper insights into cellular diversity and metabolic function through rigorous snRNA-seq analysis.

opencc-by-4.0Jan 2022View details →

single-nucleus RNA sequencing data from female Aedes aegypti maxillary palp

Single-nucleus RNA sequencing data accompanying Herre*, Goldman* et al. (2022), "Non-Canonical Odor Coding in the Mosquito" (https://doi.org/10.1016/j.cell.2022.07.024) For further analysis see: https://github.com/VosshallLab/Younger_Herre_Vosshall2020/tree/main/snRNAseq_SupplementaryData For raw sequencing files see NCBI BioProject: PRJNA794050

opencc-by-4.0Sep 2022View details →

Exploring cell diversity and fidelity in crustacean limb regeneration using single-nucleus RNA sequencing

R objects containing datasets generated by snRNA-seq on Parhyale hawaiensis limbs. These datasets have been generated during my Phd thesis.

opencc-by-4.0Apr 2024View details →

Tumor-induced alterations in single-nucleus transcriptome of atrophying muscles

<h3>Experimental Design</h3> Syngeneic C57BL/6 mice injected with Lewis Lung Carcinoma (LLC) cells were sacrificed 16 days after tumor inoculation while they experienced moderate cachexia and loss of muscle mass and function. We investigated single-nucleus transcriptomes of the tibialis anterior (TA) muscle from tumor-bearing mice and their non-tumor-bearing controls. <h3>Protocols</h3> Mice were housed at 22°C and under 50% humidity with 12 hours of light and 12 hours of dark cycles (07:00 – 19:00) and given ad libitum access to a standard rodent chow diet and water in Koc University Animal Research Facility in accordance with institutional policies and animal care ethics guidelines. 8-12 weeks old male mice with C57BL/6 background and Lewis lung carcinoma (LLC) cells were used for tumor inoculation. LLC cells were cultured in DMEM (Sigma, no. 5796) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Invitrogen). 5 million LLC cells were injected subcutaneously over the flank while control mice received PBS only. Muscle tissues were harvested at 16 days after LLC inoculation. TA muscle samples dissected from 6 mice in each group were combined and processed together. Samples were chopped with dissection scissors and placed into a lysis buffer containing 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% NP40 in nuclease-free water. Samples were homogenized using a douncer and filtered with 70 µm and 40 µm cell strainer. After centrifugation for 5 min at 500 g at 4 °C, supernatant was discarded and nuclei were resuspended and stained with DAPI and subjected to fluorescence activated cell sorting. 10X Genomics applications were used following the manufacturer’s guidelines (Chromium Next GEM Single Cell 3ʹ Reagent Kits) to prepare the libraries, which were sequenced using the Illumina HiSeq X system. <h3>Data</h3> Sequencing data was first analyzed and filtered using Cell Ranger (v7.0) Single-Cell Software Suite provided by 10X Genomics. Data was counted and mapped with cellranger count function with --include-introns option for pre-mRNAs (mm10). Further analysis was performed using Seurat (v4.3.0) R (v4.2.2) package on R Studio (v2022.12.0), which filters nuclei, normalizes expression data, and carries out principal component analysis for clustering and Uniform Manifold Approximation and Projection (UMAP) visualization. <h3>Code</h3> You can see the code in R script file.

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Data for: Multimodal convergence in the pedunculopontine tegmental nucleus: motor, sensory, and theta-frequency inputs influence the activity of single neurons

The pedunculopontine tegmental nucleus of the brainstem (PPTg) has extensive interconnections and neuronal-behavioural correlates. It is implicated in movement control and sensorimotor integration. We investigated whether single neuron activity in freely moving rats is correlated with components of skilled forelimb movement and whether individual neurons respond to both motor and sensory events. We found that individual PPTg neurons showed changes in firing rate at different times during the reach. This type of temporally specific modulation is like activity seen elsewhere in voluntary movement control circuits, such as the motor cortex, and suggests that PPTg neural activity is related to different specific events occurring during the reach. In particular, many neuronal modulations were time-locked to the end of the extension phase of the reach, when fine distal movements related to food grasping occur, indicating strong engagement of PPTg in this phase of skilled individual forelimb movements. In addition, some neurons showed brief periods of apparent oscillatory firing in the theta range at specific phases of the reach-to-grasp movement. When movement-related neurons were tested with tone stimuli, many also responded to this auditory input, allowing for sensorimotor integration at the cellular level. Together, these data extend the concept of the PPTg as an integrative structure in the generation of complex movements, by showing that this function extends to the highly coordinated control of the forelimb during skilled reach to grasp movement and that sensory and motor-related information converges on a single neuron, allowing for direct integration at the cellular level.

opencc-zeroApr 2024View details →

opencc-by-4.0Jul 2024View details →

single-nucleus RNA sequencing data of Minimal Change Disease and Focal Segmental Glomerulosclerosis patients

single-nucleus RNA sequencing data of Minimal Change Disease and Focal Segmental Glomerulosclerosis patients

opencc-by-4.0Jun 2019View details →

Analysis of patient-specific stimulation with segmented leads in the subthalamic nucleus

Supporting information for PLOS ONE article titled "Analysis of patient-specific stimulation with segmented leads in the subthalamic nucleus" by Nguyen et al. (2019)

INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS CORPUS

Intervertebral disc degeneration (IDD) arises from an intricate imbalance between the anabolic and catabolic processes governing the extracellular matrix (ECM) within the disc. Biochemical processes are complex, redundant and feedback-looped, thus improved integration of knowledge is needed. To address this, a literature-based regulatory network model (RNM) for nucleus pulposus cells (NPC) is proposed, representing the normal state of the intervertebral disc (IVD) cells, in which proteins are represented by nodes that interact among each other through activation and/or inhibition edges. This model includes 33 different proteins and 153 edges by incorporating critical biochemical interactions in IVD regulation, tested in vivo or vitro in human and animal NPC, alongside non-tissue-specific protein-protein interactions. We used the network to calculate the dynamic regulation of each node through a semi-quantitative method. The basal steady state successfully represented the activity of a normal NPC, and the model was assessed through the published literature, by replicating two independent experimental studies in human normal NPC. Pro-catabolic or pro-anabolic shifts of the network activated by nodal perturbations could be predicted. Sensitivity analysis underscored the significant influence of transforming growth factor beta (TGF-β) and interleukin-1 receptor antagonist (IL-1Ra) on the regulation of structural proteins and degrading enzymes within the system. Given the ongoing challenge of elucidating the mechanisms that drive ECM degradation in IDD, this unique IVD RNM holds promise as a tool for exploring and predicting IDD progression, shedding light on IVD phenotypes and guiding experimental research efforts.

opencc-by-4.0Jun 2024View details →

Comprehensive Chromatome Profiling Reveals Hundreds of Metabolic Enzymes in the Nucleus

Data behind figures for Comprehensive Chromatome Profiling Reveals Hundreds of Metabolic Enzymes in the Nucleus paper

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Data from: No evidence for sex differences in the electrophysiological properties and excitatory synaptic input onto nucleus accumbens shell medium spiny neurons

Sex differences exist in how the brain regulates motivated behavior and reward, both in normal and pathological contexts. Investigations into the underlying neural mechanisms have targeted the striatal brain regions, including the dorsal striatum and nucleus accumbens core and shell. These investigations yield accumulating evidence of sexually different electrophysiological properties, excitatory synaptic input, and sensitivity to neuromodulator/hormone action in select striatal regions both before and after puberty. It is unknown whether the electrical properties of neurons in the nucleus accumbens shell differ by sex and whether sex differences in excitatory synaptic input are present before puberty. To test the hypothesis that these properties differ by sex, we performed whole-cell patch-clamp recordings on male and female medium spiny neurons (MSNs) in acute brain slices obtained from prepubertal rat nucleus accumbens shell. We analyzed passive and active electrophysiological properties, and miniature EPSCs (mEPSCs). No sex differences were detected; this includes those properties, such as intrinsic excitability, action potential after hyperpolarization, threshold, and mEPSC frequency, that have been found to differ by sex in other striatal regions and/or developmental periods. These findings indicate that, unlike other striatal brain regions, the electrophysiological properties of nucleus accumbens shell MSNs do not differ by sex. Overall, it appears that sex differences in striatal function, including motivated behavior and reward, are likely mediated by other factors and striatal regions.

opencc-zeroFeb 2023View details →

opencc-by-3.0Jun 2023View details →

Data for: Representation of rewards differing in their hedonic valence in the caudate nucleus correlates with the performance in a problem-solving task in dogs (Canis familiaris)

Abstract We have investigated dogs’ (Canis familiaris) abilities in associating different sounds with food rewards of different incentive value. The establishment of the association was tested in a problem-solving behavioural paradigm, as well as in an fMRI study on the same subjects (N=20). The aim was to show behavioural, as well as parallel neural effects of the association formation between the two sounds and two different associated food rewards. The latency of solving the problem was considered as an indicator of motivational state. In our behaviour study we found that dogs were quicker in solving a problem upon hearing the sound associated with food higher in reward value, suggesting that they have successfully associated the sounds with the corresponding food value. In the fMRI study, the cerebral response to the two sounds was compared both before and after the associative training. Two bilateral regions of interest were explored: the caudate nucleus and the amygdala. After the associative training the response in the caudate nucleus was higher to the sound related to a higher reward value food than to the sound related to a lower reward value food, which difference was not present before the associative training. We found an increase in the amygdala response to both sounds after the training. In a whole-brain representational similarity analysis, we found that cerebral patterns in the caudate nucleus to the two sounds were different only after the training. Moreover, we found a positive correlation between the dissimilarity index in the caudate nucleus for activation responses to the two sounds and the difference in latencies to solve the behavioural task: the quicker the dog solved the behavioural task the greater the difference in the neural representation of the two sounds was. In summary, family dogs’ brain activation patterns reflected their expectations based on what they learned about the relationship between two sounds and their associated rewards. This dataset contains <ul> <li>Raw data (four functional runs n = 20)</li> <li>Dog brain template</li> <li>ROI results (Caudate nucleus and amygdala percentage of BOLD signal change and  caudate nucleus response to sounds during the two post-training runs adding or not the Inter-scan interval as a covariate in the GLM model n = 20,)</li> <li>RSA results ( Dissimilarity change between sounds (post-training > pre-training) and dissimilarity index per participant n = 20)</li> </ul>

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Genetic diversity and population structure from a Peruvian nucleus cattle herd using SNP data

New-generation sequencing technologies, among them SNP chips for massive genotyping, have proven to be useful for the effective management of genetic resources. Also, developing nucleus herds is an effective method for genetic improvement work. To date, molecular studies in Peruvian cattle are still in their infancy. To close this gap, we here employed two SNP panels (BovineHD and Bovine100K) to determine for the first time the Peruvian nucleus herd's genetic diversity and population structure that belong to INIA. This nucleus comprises Brahman (N=16), Braunvieh (N=14), Gyr (N=11), and Fleckvieh (N=22) breeds. Additionally, samples from a locally adapted creole cattle, the Arequipa Fighting Bull (AFB, N=12), were incorporated into the study. The genetic diversity indices in all breeds showed a high proportion of polymorphic SNPs, varying from 69.37% in Gyr to 80.81% in Braunvieh. Also, Braunvieh possessed the highest observed heterozygosity (0.53±0.17), while Brahman possessed the lowest (0.44±0.10), indicating that the former is more diverse compared to the other cattle breed groups. According to the molecular variance analysis, 83.92% of the variance occurs within individuals, whereas 16.0% occurs between populations. The pairwise FST estimates between breeds showed values that ranged from 0.054 (Braunvieh vs AFB) to 0.266 (Brahman vs AFB). Pairwise Reynold's distance showed a pattern similar to the one obtained with the FST statistics, with values ranging from 0.058 to 0.309. A dendrogram was constructed using the Neighbor-Joining clustering algorithm, and similar to the principal coordinate analysis, three groups were identified. Results showed a clear separation between Bos indicus (Brahman and Gyr) and B. taurus breeds (Braunvieh and Fleckvieh). For Fleckvieh and Braunvieh, there were two subgroups each one of them grouping with the AFB group. Similar results were obtained with ADMIXTURE analysis with K= 3 as the most optimal number for the inferred genetic structure of the populations. The results from the current study would contribute to the appropriate management avoiding loss of genetic variability in these breeds and to future improvements for this nucleus. Additional work is needed to speed up the breeding process in the Peruvian cattle system.

opencc-zeroMar 2023View details →

trajectory 61 ice nucleus in supercooled water tip4p/ice

Gromacs MD trajectories of the evolution of ice nuclei in supercooled water with the tip4p/ice model. Also included center of mass of the nucleus (length units in nm) in long range corrections to the pressure (in bar).

trajectory 53 ice nucleus in supercooled water tip4p/ice

Gromacs MD trajectories of the evolution of ice nuclei in supercooled water with the tip4p/ice model. Also included center of mass of the nucleus (length units in nm) in long range corrections to the pressure (in bar).

trajectory 65 ice nucleus in supercooled water tip4p/ice

Gromacs MD trajectories of the evolution of ice nuclei in supercooled water with the tip4p/ice model. Also included center of mass of the nucleus (length units in nm) in long range corrections to the pressure (in bar).

trajectory 67 ice nucleus in supercooled water tip4p/ice

trajectory 72 ice nucleus in supercooled water tip4p/ice

Gromacs MD trajectories of the evolution of ice nuclei in supercooled water with the tip4p/ice model. Also included center of mass of the nucleus (length units in nm) in long range corrections to the pressure (in bar).

Xenon's sedative effect is at least partly mediated by interaction with the cyclic nucleotide-binding domain (CNBD) of HCN2 channels expressed by thalamocortical neurons of the ventrobasal nucleus in mice

Xenon (70%xenon, 30%O2) application in the last 10 min of the open field test manage to sedate wild-type mice but not HCN2EA. Wild-type mice in the xenon_wild-type video can be seen moving freely in the early minutes but the general activity starts to decrease sharply in the last 2 minutes and is absent between min 9 and 10 while HCN2EA mice in the xenon_HCN2EA can be seen moving throughout the entire time of the xenon gas mixture application.