title: GSM6850215: BM_Control; Mus musculus; RNA-Seq
library_construction_protocol: Femur and tibiae were collected from 2 mice and flushed with 5mL of cold complete media (RPMI +Glutamax, 10% fetal bovine serum (FBS), 1% PenStrep) per mouse. Bone marrow was subsequently passed through a 70µm cell strainer, and collected cells were washed in 25mL of complete media and centrifuged at 600g for 4 minutes at 4˚C. Red blood cell lysis was then performed in 3mL of ACK Lysis Buffer (A1049201, Gibco) for 3-5 minutes at room temperature. Cells were washed with 25mL complete media and cryopreserved at -80˚C in freezing medium containing 15% DMSO, 20% FBS and 65% complete media at a final concentration of 1.0 x 107 cells/mL. Barcoding of individual biological samples was performed as described by McGinnis et.al.(2019). Thawed cells were suspended in 150 μLs solution containing a 1:1 molar ratio (200nM) of anchor and barcode oligonucleotide containing a unique sequence for each sample to be processed. Samples were incubated for 10 minutes at room temperature with gentle mixing every 3-5 minutes. Next, a co-anchor (200nM) was added to stabilize the barcodes within the cell membrane and incubated for additional 5 minutes. Cells were washed twice in PBS,  cell counts were measured using a Countess(TM) automated counter (Thermofischer Scientific, Burlington, ON, Canada), and viability was measured based on the ratio of cells staining with trypan blue (Thermofischer Scientific, Burlington, ON, Canada). An equal number of cells from each sample was pooled and 1000 cells/μL were further processed using the 10x Genomics Chromium scRNA-seq 3' v3 kit.