GSM9948643: GCV Exptl D5 8733; Mus musculus; RNA-Seq
A large incision was made to expose the surgical area (~2 mm on each side of the fracture) and the surrounding muscle was removed after isolating the femur.  The callus tissue was then scraped from the bone and then minced mechanically using a scalpel blade and placed in a digestion buffer (DMEM HG, Pronase, Collagenase,FBS) for 1 hour at 37 degrees celsius. During the digestion, the tube was taken out of the water bath every 15 minutes for agitation using a pipette, and was shaken every few minutes in between. The cells were then centrifuged at 200xg for 5 minutes at room temperature. The medium was aspirated and the cells were resuspended in PBS/0.01% BSA and spun again for 5 minutes. The medium was aspirated and the cells were resuspended in a MACS buffer. MACS was used to collect live cells. These cells were suspended in PBS/BSA and sent for sequencing after counting/spinning one more time to remove the MACS buffer. The library was prepared according to the manufacturer's instructions (10X Genomics, Single Cell 3' v3 protocol).