title: GSM6204160: Quiescent Pax7+ Satellite cells from Cxcr4 KO rep 1; Mus musculus; RNA-Seq
library_construction_protocol: Isolation of bulk satellite cells from hind limbs was performed as described previously (Arpke et al. 2013; Arpke and Kyba 2016). Briefly, hind limb muscles were carefully dissected and cut longitudinally with a razor blade parallel to the muscle fibers, using forceps each time pressing the blade to separate the fibers. The minced muscle was incubated shaking for 75 min in 0.2% collagenase type II (Gibco, Grand Island, NY) in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 4 mM L-glutamine 4,500 mg/L glucose, and sodium pyruvate (HyClone, Logan, UT) supplemented with 1% Pen/Strep (Gibco) at 37oC. Samples were washed twice with Rinsing Solution (F-10+), Ham's/F-10 medium (HyClone) supplemented with 10% Horse Serum (HyClone), 1% 1 M HEPES buffer solution (Gibco), and 1% Pen/Strep. Samples were centrifuged at 500 G for 5 minutes. After aspiration of supernatant, the sample was resuspended in F-10+ containing collagenase type II and dispase (Gibco), vortexed, and incubated shaking at 37⁰C for 30 min. Samples were vortexed again, drawn and released into a 10 mL syringe with a 16-gauge needle four times, then with a 18-gauge needle four times to release the cells from the muscle fibers prior to passing the cell suspension through a 40-µm cell strainer (Falcon, Hanover Park, IL). The sample was drawn and released into a 10 mL syringe with the 18-gauge needle four additional times and passed through a new 40-µm cell strainer. Samples were centrifugated for 5 min 500 G at 4 oC, and resuspended in Fluorescent-activated Cell Sorting (FACS) staining medium: Phosphate Buffered Saline (PBS, Corning, Manassas, VA) containing 2% fetal bovine serum (HyClone) and 0.5 μg/mL propidium iodide, for FACS analysis and sorting on a FACSAriaII (BD Biosciences, San Diego, CA) Libraries were prepared from total RNA from FACS sorted cells using the NEBNext Ultra Kit by GeneWiz (South Plainfield, NJ).