GSM5983931: young; Mus musculus; RNA-Seq
Two soft calluses were dissected from the fractured tibiae at  day 10 of young or aged mice and pooled as a sample, cut into small pieces (<1 mm3 62 ) and digested in 10 ml of Accumax solution (STEMCELL, 1 hour, room temperature). Cells were passed through a 35um-filter and red blood cells were lysed with ammonium chloride (5 minutes, room temperature). Cells were resuspended in staining medium (PBS with 2% fetal bovine serum) and stained with APC-anti-CD45 (Biolegend, clone 30-F11), FITC-anti-CD31 (eBioscience, clone 390), PerCP/Cy5.5-anti Ter119 (Biolegend, clone TER-119) antibodies and Dapi. Callus SSPC cells (CD45-CD31-Ter119-Dapi-) were sorted (85-micron nozzle) by FACS with a BD Aria II Instrument. Cells were loaded onto a Chromium chip (10X Genomics) followed by  encapsulation in a lipid droplet (Single Cell 3′ kit, 10X Genomics) to generate cDNA and library  according to the manufacturer's protocol. cDNA libraries were sequenced to an average of 100,000 reads per cell using Illumina Nextseq 500.