title: GSM8017442: Pulse; Mus musculus; RNA-Seq
library_construction_protocol: For isolation of stromal cells from endosteum niche, the femur and tibia bones were dissected out with the removal of muscle, connective tissue including periosteum, the epiphysis ends, the growth plates. 3mm length of the metaphysis ends of distal femur and proximal tibia were collected for future use. While the 3mm ends of proximal femur and distal tibia were removed and discarded. Bone marrow from the diaphysis was gently flushed out with 4ml flushing medium (M-199 supplemented with 10% FBS) for each bone with a 27-G needle at a fluid flow rate of 20ml/min. The rest of diaphysis middle shaft bones together with the 3mm metaphysis ends were cut into pieces (4-5 pieces for each bone), then were transferred into a 15ml centrifuge tube. The bone pieces were added with 5ml of freshly made digestion medium (1 mg/mL STEMxyme1 and 1 mg/mL Dispase II dissolved in M-199 with 2% FBS and filter sterilized with 0.45um filter) and digested on a shaker at a speed of 110rpm for 25min at 37℃. After the digestion, cell pellets were washed with 10ml cold washing buffer and passed through 70um cell strainer. Cells were then centrifuged at 300rpm for 5min, and were re-suspended in appropriate volume of cold sorting buffer (HBSS supplemented with 25mM HEPES pH7 and 2% FBS, filter sterilize using 0.2uM filter) to make the cell concentration at 0.8-1.2×107/ml. A MoFlo Astrios Sorter was used for sorting. Bright dTomato+ (0.01% of total in the pulse sample, 0.29% of total in the chase sample) cells were collected for single cell RNA-seq. scRNA-seq libraries were constructed with bone marrow mesenchymal cells from endosteal niche of pulse versus chase sample with the Single Cell 3' Reagent Kits v3.1 (10x Genomics).