GSM9628920: MSCs, TAC 4w, gene expression, biol rep 3; Mus musculus; RNA-Seq
Tibiae, femurs, and pelvis were harvested from mice (3 sham-operated and 3 TAC mice) and processed separately for isolation of MSCs. Bone marrow was first collected by flushing with cell suspension medium (PBS containing 2% FBS) and digested with collagenase type II (1 mg/mL) and DNase I (25 U/mL) in HBSS for 30 min at 37 °C. Remaining bone fragments were then crushed and further digested with collagenase type I (2.5 mg/mL) and DNase I (25 U/mL) in HBSS for 45 min at 37 °C. Cell suspensions were washed, filtered through a 100 µm strainer, and subjected to red blood cell lysis using PharmLyse solution. Cells were depleted of hematopoietic and endothelial populations using Mouse CD45 MicroBeads, CD31 MicroBeads, and Lineage Cell Depletion Kit (Miltenyi Biotec) via autoMACS separation. The negatively selected cells were washed and maintained in FACS buffer supplemented with actinomycin D (2 µM). Cells were stained with antibodies against lineage markers (Ter119, CD45, CD71, CD144, CD31, CD41, CD61, CD11b, Gr1, CD45R, CD5) and Leptin receptor (Lepr). Lin-Lepr+ cells were sorted as MSCs. RNA-seq libraries were prepared using a NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's protocols.