title: GSM5059687: Aged MuSCs 7dpi; Mus musculus; RNA-Seq
library_construction_protocol: For tissue collection, mice were anesthetized with 3% isoflurane, then euthanized by cervical dislocation, bilateral pneumothorax and removal of the heart. Hind limb muscles (tibialis anterior and gastrocnemius) of control and experimental mice were quickly harvested using sterile surgical tools and placed in separate plastic petri dishes containing cold PBS. Using surgical scissors, muscle tissues were minced and transferred into 50 mL conical tubes containing 20 mL of digest solution (2.5 U/mL Dispase II and 0.2% [~5,500 U/mL] Collagenase Type II in DMEM media per mouse). Samples were incubated on a rocker placed in a 37oC incubator for 60 min with manual pipetting the solution up and down to break up tissue every 30 minutes using an FBS coated 10 mL serological pipette. Once the digestion was completed, 20 mL of F10 media containing 20% heat inactivated FBS was added into each sample to inactivate enzyme activity. The solution was then filtered through a 70 µm cell strainer into a new 50 mL conical tube and centrifuged again at 350xg for 5 min. The pellets were re-suspended in 6 mL of staining media (2% heat inactivated FBS in Hank's Buffered Salt Solution - HBSS) and divided into separate FACS tubes. The FACS tubes were centrifuged at 350xg for 5 min and supernatants discarded. The cell pellets were then re-suspended in 200 µL of staining media and antibody cocktail containing Sca-1:APC (1:400), CD45:APC (1:400), CD11b:APC (1:400), Ter119:APC (1:400), CD29/b1-integrin:PE (1:200), and CD184/CXCR-4: BIOTIN (1:100) and incubated for 30 minutes on ice in the dark. Cells and antibodies were diluted in 3mL of staining solution, centrifuged at 350xg for 5 min, and supernatants discarded. Pellets were resuspended in 200uL staining solution containing PECy7:STREPTAVIDIN (1:100) and incubated on ice for 20 minutes in the dark. Again, samples were diluted in 3mL staining solution, centrifuged, supernatants discarded, and pellets re-suspended in 200uL staining buffer. Live cells were sorted from the suspension via addition of 1 µg of propidium iodide (PI) stain into each experimental sample and all samples were filtered through 70 μm cell strainers before the FACS. Cell sorting was done using a BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA) and APC negative, PE/PECy7 double-positive MuSCs were sorted into staining solution for immediate processing. Freshly isolated MuSCs were sorted into staining solution, enumerated by hemocytometer, and re-suspended into PBS. Cells were loaded into the 10x Genomics chromium single cell controller for each time point and age group were captured into nanoliter-scale gel bead-in-emulsions (GEMs). cDNAs were prepared using the single cell 3′ Protocol as per manufacturer's instructions and sequenced on a NextSeq 500 instrument (Illumina) or NovaSeq instrument (Illumina) with 26 bases for read1 and 98 bases for read2.