Extraction and sequencing of single nuclei from murine skeletal muscles.
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ABSTRACT:
SUBMITTER: Santos MD
PROVIDER: S-EPMC8339382 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Single-cell RNA sequencing (scRNA-seq) is a powerful technique for deconvoluting and clustering thousands of otherwise intermingled cells based on their gene expression. Here, we present a complete protocol for the unbiased evaluation of regenerating murine skeletal muscle using scRNA-seq. The skeletal muscle is unique in its cellular composition as being primarily multinucleated muscle cells (myofibers). This protocol focuses on isolating mononuclear cells from muscle for subsequent scRNA-seq analysis and can be modified to assess cell populations in other tissues of interest. For complete details on the use and execution of this protocol, please refer to Liu et al. (2015) and Oprescu et al. (2020).
| S-EPMC7580090 | biostudies-literature
Project description:It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.
| S-EPMC3856806 | biostudies-literature
Project description:Muscleblind-like 1 (MBNL1) is an alternative splicing factor involved in postnatal development of skeletal muscles and heart in humans and mice, and its deregulation is known to be pivotal in the onset and development of myotonic dystrophy (DM). In fact, in DM patients this protein is ectopically sequestered into intranuclear foci, thus compromising the regulation of the alternative splicing of several genes. However, despite the numerous biochemical and molecular studies, scarce attention has been paid to the intranuclear location of MBNL1 outside the foci, although previous data demonstrated that in DM patients various splicing and cleavage factors undergo an abnormal intranuclear distribution suggestive of impaired RNA processing. Interestingly, these nuclear alterations strongly remind those observed in sarcopenia i.e., the loss of muscle mass and function which physiologically occurs during ageing. On this basis, in the present investigation the ultrastructural localization of MBNL1 was analyzed in the myonuclei of skeletal muscles from healthy and DM patients as well as from adult and old (sarcopenic) mice, in the attempt to elucidate possible changes in its distribution and amount. Our data demonstrate that in both dystrophic and sarcopenic muscles MBNL1 undergoes intranuclear relocation, accumulating in its usual functional sites but also ectopically moving to domains which are usually devoid of this protein in healthy adults. This accumulation/delocalization could contribute to hamper the functionality of the whole splicing machinery, leading to a lower nuclear metabolic activity and, consequently, to a less efficient protein synthesis. Moreover, the similar nuclear alterations found in DM and sarcopenia may account for the similar muscle tissue features (myofibre atrophy, fibre size variability and centrally located nuclei), and, in general, for the aging-reminiscent phenotype observed in DM patients.
| S-EPMC3794341 | biostudies-literature
Project description:Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.
| S-EPMC4807405 | biostudies-literature
Project description:BACKGROUND:The development of skeletal muscle is closely related to the efficiency of meat production and meat quality. Chicken skeletal muscle development depends on myogenesis and adipogenesis and occurs in two phases-hyperplasia and hypertrophy. However, cell profiles corresponding to the two-phase muscle development have yet to be determined. Single-cell RNA-sequencing (scRNA-seq) can elucidate the cell subpopulations in tissue and capture the gene expression of individual cells, which can provide new insights into the myogenesis and intramuscular adipogenesis. RESULTS:Ten cell clusters at the post-hatching developmental stage at Day 5 and seven cell clusters at the late developmental stage at Day 100 were identified in chicken breast muscles by scRNA-seq. Five myocyte-related clusters and two adipocyte clusters were identified at Day 5, and one myocyte cluster and one adipocyte cluster were identified at Day 100. The pattern of cell clustering varied between the two stages. The cell clusters showed clear boundaries at the terminal differentiation stage at Day 100; by contrast, cell differentiation was not complete at Day 5. APOA1 and COL1A1 were selected from up-regulated genes in the adipocyte cluster and found to be co-expressed with the ADIPOQ adipocyte marker gene in breast muscles by RNA in situ hybridization. CONCLUSIONS:This study is the first to describe the heterogeneity of chicken skeletal muscle at two developmental stages. The genes APOA1 and COL1A1 were identified as biomarkers for chicken intramuscular fat cells.
| S-EPMC7603756 | biostudies-literature
Project description:Data Access Committee EGAC00001002603
| EGAC00001002603 | EGA
Project description:We have previously documented a time-course of sarcopenia development in female C57Bl/6J mice, with significant loss of quadriceps muscle mass by 24 m that is more pronounced by 27-29 m. The present study used gastrocnemius muscles from female C57Bl/6J mice aged 3, 15, 24 and 29 months to investigate the expression of genes that coincide with, or potentially precede, muscle loss. The microarray analyses of ageing gastrocnemius muscle indicated that the Functional Theme Protein catabolism was first discernable in 15 and 24 age contrast and then again in the age contrast between 24 m and 29 m. A common Functional Theme in all age contrasts from the microarray analysis was extracellular matrix suggesting continual remodeling of skeletal muscle during ageing. Metabolic traits, particularly those involving altered fatty acid and glucose metabolism were apparent in the contrast between 24 m and 29 m. This may reflect inefficiency of lipid metabolism with ageing caused by altered mitochondrial function, which has been implicated in ageing processes in other studies. There were no functional themes associated with oxidative stress, inflammation or immune cell infiltration in any age contrast, likely indicating that these factors are not primary drivers of sarcopenia. Ageing was also associated with increased global gene expression variance, consistent with generalised decreased control of gene regulation.
2014-01-10 | GSE53959 | GEO
Project description:We have previously documented a time-course of sarcopenia development in female C57Bl/6J mice, with significant loss of quadriceps muscle mass by 24 m that is more pronounced by 27-29 m. The present study used gastrocnemius muscles from female C57Bl/6J mice aged 3, 15, 24 and 29 months to investigate the expression of genes that coincide with, or potentially precede, muscle loss. The microarray analyses of ageing gastrocnemius muscle indicated that the Functional Theme Protein catabolism was first discernable in 15 and 24 age contrast and then again in the age contrast between 24 m and 29 m. A common Functional Theme in all age contrasts from the microarray analysis was extracellular matrix suggesting continual remodeling of skeletal muscle during ageing. Metabolic traits, particularly those involving altered fatty acid and glucose metabolism were apparent in the contrast between 24 m and 29 m. This may reflect inefficiency of lipid metabolism with ageing caused by altered mitochondrial function, which has been implicated in ageing processes in other studies. There were no functional themes associated with oxidative stress, inflammation or immune cell infiltration in any age contrast, likely indicating that these factors are not primary drivers of sarcopenia. Ageing was also associated with increased global gene expression variance, consistent with generalised decreased control of gene regulation. Microarray profiling was performed in gastrocnemius muscles from female C57Bl/6J mice aged 3, 15, 24 and 29 months.
2014-01-10 | E-GEOD-53959 | biostudies-arrayexpress
Project description:Next-generation sequencing provides an opportunity for an in-depth biocomputational analysis to identify gene expression patterns between soleus and tibialis anterior, two well-characterized skeletal muscles, and analyze their gene expression profiling. RNA read counts were analyzed for differential gene expression using the R package edgeR. Differentially expressed genes were filtered using a false discovery rate of less than 0.05 c, a fold-change value of more than twenty, and an association with overrepresented pathways based on the Reactome pathway over-representation analysis tool. Most of the differentially expressed genes associated with soleus are coded for components of lipid metabolism and unique contractile elements. Differentially expressed genes associated with tibialis anterior encoded mostly for glucose and glycogen metabolic pathway regulatory enzymes and calcium-sensitive contractile components. These gene expression distinctions partly explain the genetic basis for skeletal muscle specialization, and they may help to explain skeletal muscle susceptibility to disease and drugs and further refine tissue engineering approaches.
| S-EPMC8621074 | biostudies-literature
Project description:BackgroundMitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles.Methodology/principal findingsAdult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca(++) retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor.Conclusion/significanceWe conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor.
| S-EPMC3467208 | biostudies-literature