GSM8502288: Mouse periosteal cell replicate 2, scRNA-seq; Mus musculus; RNA-Seq
To isolate periosteal cells, the dissected femurs and tibias from C57BL6 WT mice were placed in PBS, and the overlying skin, fascia, and muscle were carefully removed. The bones with periosteum were incubated in ice-cold PBS with 1% FBS for 30min, and the loosely associated periosteum was peeled off using scalpel, forceps, and dissecting scissors. The soft floating periosteal tissues were collected and incubated with 5-10mL of 0.2% collagenase and 10% FBS in PBS at 37ºC for 1 hour and the dissociated periosteal cells were washed with PBS, filtered with a 40um strainer, and resuspended at 1x10^7cells/mL. Isolated cells were FACS-sorted based on CD45, CD31, Ter-119, and DAPI staining. Trypan blue stain was used to assess cell viability and number. Cells were then sorted in 10% FBS with MEM-alpha media. Libraries were constructed using the Chromium Single Cell 3' v3.1 (10x genomics, PN-11000121) Reagent Kit, according to the manufacturer's protocol. Amplified cDNA and final libraries were evaluated on an Agilent BioAnalyzer using a High Sensitivity DNA Kit (Agilent Technologies).