GSM5710212: BM MSC and endothelial cells - Cxcr4+/+ [CTR-1]; Mus musculus; RNA-Seq
Long bones were flushed with HBSS supplemented with 2% heat-inactivated FBS, 1% Penicillin/Streptomycin, 1% L-glutamine, 1% HEPES and 200 U/ml Collagenase IV. The flush out was digested for 30 min at 37C. Flushed bones were cut into bone chips and digested separately for 45 min at 37°C under agitation (120rpm) and washed with HBSS 2% FBS, 1% Penicillin /Streptomycin, 1% L-glutamine, 1% HEPES. Both cell suspensions were filtered through cell strainers and combined. Cells from 2 mice per genotype were pooled. Cells were stained with antibodies against Ter119, CD45, lineage (LIN) (B220, CD19, CD11b, CD3e), CD31, CD144, LEPR and CD71. CD45- Ter119- CD71- LIN- LEPR+ and CD45- Ter119- CD71- LIN- CD31+ CD144+ cells were sorted into the same tube with 350 μL DMEM with 20% FBS. The libraries were prepared using the Chromium Single Cell 3ʹ Reagent Kits v3.0 and run on an Illumina NovaSeq system with 100-bp paired-end reads to a coverage of ~ 40,000 - 44,000 mean reads per cell and ~ 80% saturation. The sequencing reads were aligned onto the Mus musculus GRCm38 (mm10) reference genome.