GSM7124094: BM_LK_Expt1_Vehicle_1; Mus musculus; RNA-Seq
Murine bone marrow cells were collected from the femora, tibiae and ilia. Red cells were lysed with ammonium chloride STEMCELL Technologies, Vancouver, Canada) and haematopoietic progenitors enriched using the EasySepTM Mouse Hematopoietic Progenitor Cell Enrichment Kit (STEMCELL Technologies).  Cells were stained with fluorophore conjugated antibodies c-Kit APC-Cy7 (Biolegend, clone 2B8, 1/200), CD150 PE-Cy7 (Biolegend, clone TC15-12F12.2 1/200), CD48 APC (eBioscience, clone HM48-1, 1/200), EPCR PE (STEMCELL Technologies, clone RMEPCR1560, 1/400), Sca1 BV421 (Biolegend, clone D7, 1/100). The EasySepTM Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies) and BV510 streptavidin (Biolegend) was used to stain lineage-positive cells. Dead cells were excluded using 7-aminoactinomycin D (Thermo Fisher Scientific). An aliquot of non-lineage- depleted cells was retained for unstained and single-stain controls. Cells were analysed and sorted on an Influx sorter (BD Biosciences); 20,000 Lineage-negative, c-kit +cells per mouse were sorted into PBS + 2% FCS for scRNA-seq analysis Cells were processed according to manufacturer's protocol using Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 reagents.