Project description:Skeletal muscle possesses the ability to adapt its size in response to milieus, which is called plasticity. Overload like resistance training induces the increment of muscle mass called muscle hypertrophy. Muscle stem cells (also known as muscle stem cells) function to supply new nuclei for myofiber during the overload in muscle. Using compensatory hypertrophy in plantaris muscles, we isolated MuSCs from plantaris muscles 4 days after surgery. Control MuSCs were also prepared from sham plantaris muscles.
Project description:We compared transcriptome of real quiescent MuSCs and freshly isolated MuSCs with in-situ fixation,FISC cultured for 24hrs,48hrs and 72hrs by RNA-sequencing.
Project description:We compared the transcriptome of isolated 0.5% paraformaldehyde-fixed MuSCs to Live (freshly isolated, not fixed) MuSCs by RNA-sequencing.
Project description:Sirt6, the NAD+-dependent deacetylase, has been described to deacetylate H3K9, H3K18, and H3K56. However, analysis of the acetylation status revealed that loss of Sirt6 caused a massive increase of histone H3K56ac levels but no detectable change of histone H3K9ac and H3K18ac, indicating that SIRT6 is the dominant deacetylase for H3K56ac in muscle stem cells (MuSCs). Further, we investigate genome-wide H3K56ac profiling in the absence of Sirt6 in MuSCs and mouse embryonic stem cells (mESCs) using high throughput sequencing (ChIP-seq).
Project description:We generated MyoD-KI mice to distinguish the heterogenous MuSCs populations based on MYOD expression. Using the mice, we performed RNA-seq to identify the unique gene expression profile in the heterogenous MuSCs population.
Project description:We report the application of single myofiber ATAC-Seq (smfATAC-Seq) to investigate the chromatin accessibility of a single myofiber without the presence of confounding muscle resident cell types. This method demonstrates that open chromatin regions of myonuclei can be tagmentated and high-quality sequencing ready libraries can be generated from these fragments. To perform comparative analysis as well as to demonstrate the applicability of the smfATAC-Sew to study changes in chromatin of myonuclei within different contexts, smfATAC-Seq was performed both on uninjured myofibers as well as injured myofibers seven days after induced injury. Furthermore, ATAC-Seq on 5000 muscle stem cells (MuSCs) was also performed according to the previously described OMNI ATAC-Seq protocol (Corces, M.R. et al. Nature Methods, 2017) in order to compare the sequencing quality of the smfATAC-Seq as well as to demonstrate the changes in open chromatin that occur from the stem cell state to the fully differentiated myofibers. smfATAC-seq resulted in comparable coverage and sequencing depth to the ATAC-seq performed on MuSCs and allowed for peak calling and differential peak analysis. These analysis revealed that the open chromatin state of uninjured and injured myofibers after seven days are mostly similar although some regions invovled in immune response remain in an open state in the injured myofibers compared to the uninjured myofibers and the regions involved in structural formation of the muscle are more accessible in the case of regeneration. Even though certain differences in the open chromatin are observed, smfATAC-Seq analysis suggest that overall, the open chromatin state of the myonuclei returns back to homeostasis after seven days of regeneration. Furthermore, smfATAC-Seq comparison with the ATAC-Seq from MuSCs show the differences in open chromatin regions between these conditions. Increased accessibility of genes involved in myogenesis and structural components can be observed in the myofibers compared to MuSCs while MuSCs show increased accessibility in regions involved in membrane permeability and signalling pathways. In addition, the regions that are accessible for both conditions include genes involved in mitochondrial transport, regulation of transcription and regulation of metabolites and energy. Overall, this study introduces smfATAC-Seq that succesfully assesses the genome-wide chromatin accessibility of a single myofiber with relatively high sequencing depth. The smfATAC-Seq can be used to perform comparative analysis between different conditions such as injury. However, this method can be readily applied to study differences between young and old myofibers or in the context of muscular dystrophy, cachexia, and exercise.
