GSM6732986: Aged+GB; Mus musculus; RNA-Seq
GA muscles were isolated from mice immediately following euthanasia. The muscles were minced and homogenized with homogenization buffer, 0.25M sucrose and 1% BSA diluted in Mg2+-free, Ca2+-free, RNase-free PBS (#AM9624, Invitrogen, Grand Island, NY, USA). The homogenates were incubated with 2.5% Triton-X100/RNase-free PBS at a 1:6 ratio for 5 minutes. After filtering through a 100-μm strainer, samples were centrifuged at 3000 × g for 10 min at 4 °C, resuspended in 2% BSA/RNase-free PBS, and then filtered through a 40-μm strainer again. Nuclei were labeled by DAPI with 0.2 U/µL RNase inhibitor (#N8080119, Thermo Fisher Scientific). Labeled nuclei were sorted by FACS Aria II Cell Sorter with 70 μm nozzle (BD Biosciences, San Jose, CA, USA) into 2% BSA/RNase-free PBS with RNase inhibitor. Isolated nuclei were observed by confocal fluorescent microscope (Leica TCS SP8, Wetzlar, Germany) to confirm the integrity. To adjust the optimal concentration of nuclei for the 10X Chromium system, isolated nuclei were counted and diluted or concentrated by centrifugation at 250 × g for 5 min at 4 °C. Single-nucleus RNA-sequencing libraries were constructed by the Chromium Single Cell 3' reagent kit v2 (10X Genomics, Pleasanton, CA, USA) per the manufacturer's protocol. Following the library preparation, the libraries were sequenced on the NovaSeq 6000 System (Illumina, San Diego, CA, USA)