GSM4626772: Bone marrow-derived stromal cells; Mus musculus; RNA-Seq
The left femurs of 5 Diversity Outbred mice (4 females, 1 male) were isolated and cleaned thoroughly of all muscle tissue followed by removal of the distal epiphysis. For each femur, marrow was exuded by centrifugation at 2000g for 30 seconds into a sterile tube containing 35ul freezing media (90% FBS, 10% DMSO). The marrow was then triterated 6 times on ice after addition of 150ul ice cold freezing media and again after further addition of 1ml ice cold freezing media until no visible clumps remained prior to being placed into a Mr. Frosty Freezing Container (Thermo Scientific) and stored overnight at -80C. Samples were transferred the following day to liquid nitrogen for long term storage. Day 10 mineralized bone marrow cultures were washed twice with DPBS followed by addition of 0.5ml 60mM EDTA for a 15-minute room temperature incubation. The cultures were aspirated and solution replaced for a second incubation. Cultures were then washed with 0.5ml HBSS followed by a 37C shaking incubation containing 0.5ml 8mg/ml collagenase in HBSS/4mM CaCl 2 for 10 minutes after which cultures were triterated 10 times, incubated at 37C for 20 minutes and transferred to 1.5ml tubes. Cells were then pelleted in Sorvall tabletop centrifuge at 500g for 5 minutes at room temperature, resuspended into 0.5ml 0.25% trypsin and incubated for 15 minutes at 37C followed by triteration and incubation for an additional 15 minutes after which 0.5ml media was added, cells were re-triterated, re-pelleted, resuspended into 0.5ml bone marrow differentiation media (Alpha MEM, 10% FBS, 1% Penicillin Streptomycin, 0.01% Glutamax, 50mg/ml Ascorbic Acid, 1M B-glycerophosphate, 100uM Dexamethasome) and counted. The samples were pooled and concentrated to 800 cells/ul in sterile PBS supplemented with 0.1% BSA. The single cell suspension was loaded into a 10xChromium Controller (10X Genomics, Pleasanton, CA, USA), aiming to capture 8,000 cells, with the Single Cell 3' v2 reagent kit, according to the manufacturer's protocol. Following Gem capturing and lysis, cDNA was amplified (13 cycles) and the manufacturer's protocol was followed to generate sequencing library.
GEO Accession
GSM4626772