GSM5209369: LS005: MPLW515 BM stroma3; Mus musculus; RNA-Seq
Hindlimb bones were dissected and central marrow tissue was carefully flushed to minimize tissue disruption using FACS buffer (HBSS + 2% FBS). Intact bone marrow plugs were digested with a mixture of Collagenase IV (200 U/ml, Worthington), Liberase DH (0.3 Wünsch units/ml, MilliporeSigma), and DNase I (200 U/ml, MilliporeSigma) diluted in HBSS (with Ca2+ and Mg2+) at 37ºC for 25 minutes. Samples were then dissociated by repetitive pipetting with a P1000 tip and filtered through a 40 µm cell strainer. Immunostaining was performed for 30 minutes on ice with anti-CD45 (30F-11) and anti-Ter119 to exclude hematopoietic cells. Incubation with Calcein Violet-AM was used to stain live cells and exclude debris. SYTOX Green was added shortly before FACS analysis to exclude dead/dying cells. FACS-isolated stromal cells (CD45/Ter119/GFP/SYTOX Green negative, Calcein Violet-AM positive) were processed using Chromium Controller (10x Genomics). scRNA-seq libraries were constructed using Chromium Single Cell 3' v3 Reagent Kit according to manufacturer instructions. Sequencing was performed on an Illumina NovaSeq 6000.