GSM6820991: bone_calcitriol_scRNA; Mus musculus; RNA-Seq
The femurs were pooled for each group and treated as one sample. Epiphyses were cut off, and the marrow was flushed out by centrifugation. Mouse femurs were disrupted by Cryo-Press (Microtec Co., Ltd.) and digested with 1.6 U/mL collagenase D (Roche Diagnostics) in HBSS; pH = 7.8; FUJIFILM Wako Pure Chemical Corporation) for 30 min. The supernatant was discarded. Enzyme treatment and removal of supernatant steps were repeated twice. Residual bone pieces were treated with 20 mM ethylene glycol bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid disodium salt solution (neutral) (EGTA; Nacalai Tesque, Inc.) in HBSS for 15 min and then with 1.6 U/mL collagenase D for 20 min to collect the cells. Treatment with collagenase D was repeated twice. All digestion steps were carried out in a 3.5 mL solution in a six-well petri dish, on a rotating shaker at 120 rpm in a 37°C incubator. After centrifugation and removal of supernatant, cell pellets were suspended in 1% (w/v) BSA-PBS(-); pH = 7.0 (Nacalai Tesque, Inc.). To remove CD45-positive cells, cell suspensions were mixed with CD45 microbeads (Miltenyi Biotec) and incubated at 4°C for 15 min in PEB buffer and rinsed. PEB buffer contains MACS® BSA stock solution (Miltenyi Biotec) and autoMACS rinsing solution (Miltenyi Biotec) in a 1:20 ratio. Cell suspensions were passed through the LD column (Miltenyi Biotec). The flow-through was collected, centrifuged, and resuspended in 1% BSA-PBS. Cells were passed through a 40 μm nylon strainer and used for scRNA-seq. Library was constructed according to the manufacter's instructions (Chromium Next GEM Automated Single Cell 3' Reagent Kits v3.1, 10x Genomics). Briefly, mouse bone cells were resuspended in the 1% BSA-PBS and set together with the other reagents on the Chromium Connect to generate the sequence library.