title: GSM8835955: Muscle GFP+, WT (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 (TA and GTN) of control and experimental mice were quickly harvested using sterile surgical tools and placed in separate plastic petri dishes containing cold phosphate-buffered saline (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% [~1000 U/mL] Collagenase Type II, 1 mg/mL of hyaluronidase in Dulbecco's modified Eagle medium [DMEM] per mouse). Samples were incubated on a rocker placed in a 37 ̊C incubator for 60 min with manual pipetting the solution up and down to break up tissue every 30 min using an FBS-coated 10 mL serological pipette. Once the digestion was completed, 20 mL of F12 media containing 20% heat inactivated FBS was added into each sample to inactivate enzyme activity. The solution was then filtered through a 70 mm cell strainer into a new 50 mL conical tube and centrifuged again at 300g for 5 min. 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 mm cell strainers before the FACS. Cell sorting was done using a BD FACS Aria III Cell Sorter (BD Biosciences, San Jose, CA). GFP+ PI- and GFP- PI- cells were sorted into 0.02% BSA PBS solution for immediate processing. RNA libraries for RNA Seq were prepared using GEM Single Cell 3' HT Reagent Kits v3 kits from 10X Genomics according to the manufacturer's protocols. Library preparation and next-generation sequencing was carried out in the Advanced Genomics Core at the University of Michigan. Cells were loaded into the 10X Genomics chromium single cell controller for each sample and captured into nanoliter-scale gel bead-in-emulsions. cDNAs were prepared using the single cell protocol as per manufacturer's instructions.