GSM7691509
Cells were isolated from the tibia and femur and crushed once, using mortar and pestle, then cut into small pieces with scissors. The tissue was subjected to three rounds of enzymatic digestion with 0.2%collagenaseat 37°C under agitation for 30min each. Cells were filtered through a 70-μm strainer and centrifuged at 1500 rpm for 5 minutes at 4°C.  Red blood cells were lysed using NH4Cl (StemCell Technologies, Vancouver, Canada) for 10 min on ice, washed with staining media (HBSS (Thermo Fisher Scientific, Waltham, MA, USA) containing 2% fetal bovine serum (Life Technologies: 10437-028), 1% HEPES (10 mM) (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific) and resuspended in HBSS for subsequent analyses Libraries were prepared using the Chromium single cell 3ʹ reagent v2 protocol (10x Genomics) per the manufacturer's recommendations. Sequencing was performed by the Illumina NovaSeq 6000