title: GSM8472478: Young Skull Stromal Haem; Mus musculus; RNA-Seq
library_name: GSM8472478
library_construction_protocol: Mice from each age group were euthanized and skull and femur were harvested. Skulls were chopped with scissors in FACS buffer before crushed with mortar and pestle; femurs were crushed without chopping. BM stromal samples were dissociated with Collagenase I (Gibco, Cat# 17100-017, 2 mg/ml) and Collagenase IV (Gibco, Cat# 17104-019, 2 mg/ml) in PBS for 20 minutes at 37°C with intermittent shaking. Cell suspensions were strained through a 40 mm mesh filter and washed with FACS buffer. Cells were resuspended and incubated with biotinylated rat monoclonal anti-hematopoietic lineage antibody cocktail (Miltenyi-Biotec, Cat# 130-092-613, 1:50 dilution). Cells were washed, resuspended in FACS buffer with mouse monoclonal anti-Biotin MicroBeads (Miltenyi-Biotec, Cat# 130-105-637, 1:50 dilution) and incubated before being loaded into a magnetic-associated cell sorting (MACS) column (Miltenyi-Biotec, Cat# 130-042-201) for Lin-depletion. Lin-negative cells were further incubated with rat monoclonal anti-CD45 MicroBeads (Miltenyi-Biotec, Cat# 130-052-301, 1:50 dilution), rat monoclonal anti-CD117 MicroBeads (Miltenyi-Biotec, Cat# 130-091-224, 1:50 dilution), biotinylated rat monoclonal anti-CD71 (Biolegend, Cat# 113803, 1:100). Cells were washed, resuspended in FACS buffer with mouse monoclonal anti-Biotin MicroBeads (Miltenyi-Biotec, Cat# 130-105-637, 1:50 dilution) and incubated before being loaded into a magnetic-associated cell sorting (MACS) column (Miltenyi-Biotec, Cat# 130-042-201) for further hematopoietic depletion. Single cell suspensions were processed with BD Rhapsody Express system according to the manufacturer's instructions. Then, single cell whole transcriptome libraries were prepared using BD Rhapsody WTA Reagent kit (BD Biosciences).
identifiers.namespace: GEO
identifiers.id: GSM8472478_r1