GSM8999237: Recovered mouse sample 11; Mus musculus; RNA-Seq
Primary cells were extracted from the femur and tibia of C57BL/6, Fgd5-mCherry+ mice (Jax# 027785) aged 3-6 months. Briefly, mice were euthanized, tibia and femur were extracted, cleared of the flesh, and crushed in Sample Media (Phosphate Buffer Saline - PBS) supplemented with 2% Fetal Bovine Serum and 2 mM EDTA) on ice. Bone marrow cells were then filtered through a 70um mesh. Isolation of mononuclear cells was performed using the Ficoll-PaqueTM premium 1.084 (Cytiva, cat #17544602) by loading 3 ml of pre-warmed H1083 reagent under cells resuspended in 7 ml of room temperature Sample Media. The cells were centrifuged for 30 min at 400 g without breaks at room temperature. Upon the centrifugation, the mononuclear layer was washed once with Sample Media. Cells were then stained with a combination of antibodies: Lineage Cocktail- PacBlue Cat# 133310, Sca1-APC Cat# 108112, cKit-APCcy7 Cat# 1058264. Healthy, sick and recovered Fgd5-mCherry+ mice Progenitors (Lineage-Sca1+ cKit+) were sorted, ~50,000 cells per mouse, to 500µl sample Media . Barcoded cDNA libraries were conducted by Dr. Yulia Haim of the Obesity research lab (Prof. Assaf Rudich's lab) Department of Clinical Biochemistry and Pharmacology, Ben Gurion University of the Negev, using 10x Genomics Chromium X and Next GEM Single Cell 3' Reagent Kits v3.1 following the company protocol.