GSM7732482: Con_13wk_rep2; Mus musculus; RNA-Seq
Soft tissues and periosteum were removed from freshly collected femurs and tibiae. Bone marrow was flushed out by PBS with 1% BSA. Bones were reduced in fragments and digested with sequential incubations of LiberaseTM (1 Wunsch unit/mL) in HBSS buffer and 5 mM EDTA solution in Mg2+- and Ca2+-free PBS (4 times each for 20 min at 37 ºC). Cells were resuspended in sorting buffer (2 mM EDTA in PBS with 0.5% BSA) and passed through a 70 µm strain. Biotinylated antibodies against CD45, CD45R, CD71, TER-119, CD5, CD11b, Gr-1, Ly-6B.2, and CD3e, and anti-biotin magnetic beads were used to remove hematopoietic cells through columns containing ferromagnetic spheres. Eluted cells were resuspended in PBS with 0.5% BSA, counted and used for scRNA-sequencing assays. Chromium Single Cell 3' v3 Reagent Kit (10X Genomics) was used for library preparation according to manufactur's instructions. Libraries were sequenced using a NextSeq 2000 sequencer.