GSM7372917: Akita-20wks; Mus musculus; RNA-Seq
Tibias and femurs from three mice per group were harvested with the epiphysis surgically removed. The central bone marrow was flushed out with media (M-199 with 10% FBS and 1% P/S) and discarded, followed by scraping clean the periosteum with a scalpel. The metaphysis and mid-shafts were cut into small pieces with scissor. Bone pieces were digested with the STEMxyme1 (1 mg/ml, Worthington, LS004106) and Dispase II (1 mg/ml, ThermoFisher Scientific, 17105041) for 25 minutes at 37 °C in a cell culture shaker (ThermoFisher Scientific, MAXQ 4450). The digested cells were centrifuged at 300g for 3 mins and then suspended in 200 ul staining buffer (HBSS+2%FBS). Samples were stained and FACS sorted for CD45-(Invitrogen, 12-0451-82), CD3 (Biolegend, 100205), CD45R/B220 (Biolegend, 103207), CD19-(Biolegend, 115507), Gr1- (Biolegend, 108407), CD11b-(Biolegend, 101207),Ter119--( Invitrogen, 17-5921-82), CD71-(Biolegend, 113811), 7-AAD+-( Invitrogen, 00-6993), and calcium AM+( Invitrogen, C1430) with MoFlo Astrios Sorter (Beckman coulter ). They were used to construct barcoded scRNA-seq libraries by using the Chromium single cell 3' v2 reagent kit (CG000204_ChromiumNextGEMSingleCell3'v3.1, 10x Genomics).