title: GSM8115223: M35; Mus musculus; RNA-Seq
library_construction_protocol: Isolated hindlimbs were mechanically crushed into 2mL of Hank's Balanced Salt Solution (Sigma Aldrich, H6648). Supernatant was filtered through a 70µm filter (Corning, 352350) into a 50mL Falcon tube. The remaining tissue was further incubated with 2 Wunsch units of LiberaseTM (Sigma Aldrich, 5401127001) and 0.25% Trypsin-EDTA (Fisher Scientific, 15400054) at 37°C for 30 min with agitation. The supernatant was again filtered through a 70µm filter into a 50mL Falcon tube. Cells were then stained with CD45-APC (ThermoFisher, MCD4505), Ter119-APC (ThermoFisher, 17-5921-82) and DAPI (Invitrogen, D1306) for 30min on ice. Cells were washed with FACS Buffer (Phosphate Buffered Solution plus 2% fetal bovine serum). Flow cytometry and sorting was performed with a 4-laser BD LSR II and FACSDivia. TdTomato+/DAPI-/APC- cells were sorted into FACS Buffer. RNA libraries were prepared for sequencing using standard Illumina protocols