title: GSM8772510: scRNA-seq, WT bone marrow cells; Mus musculus; RNA-Seq
library_construction_protocol: Primary BM cells were isolated from the femurs and tibias of 8-12-week-old mice. Briefly, bones were dissected, cleaned of muscle and connective tissue, and flushed with ice-cold α-MEM (Gibco) using a 25G needle into a 50mL falcon tube. The resulting cell suspension was passed through a 70 μm cell strainer (Corning) to remove debris and clumps, followed by centrifugation at 300 × g for 5 minutes at 4°C. To eliminate erythrocytes, the cell pellet was resuspended in pre-warmed RBC lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH7.4) and incubated for 2 minutes at room temperature. The reaction was quenched by adding cold α-MEM, followed by two washes with 1× PBS and centrifugation at 500 × g for 5 minutes at 4°C. Cells were then resuspended in PBS + 0.04% bovine serum albumin (BSA), filtered through a 35 μm strainer, and stained with 7-AAD viability dye (BioLegend, #420403) to exclude dead cells. Live, single cells were sorted using a Cytek Aurora 4 flow cytometer. Libraries were constructed with a Chromium Single Cell 3′ Library kit, according to the manufacturer's protocol.
identifiers.namespace: GEO
identifiers.id: GSM8772510_r1