title: scRNA-Seq of homo sapiens: posterior longitudinal ligament4
design: After thoroughly flushing the blood with saline, the collected samples were stored in MACS Tissue Storage Solution (Miltenyi, Cat# 130-100-008) at 4. Subsequently, the harvested tissues were digested using RPMI 1640 medium (Gibco, Cat#11875093) supplemented with 0.3% type 1 collagenase (Gibco, Cat# 17100017) and 0.4% dispase II (Sigma-Aldrich, Cat# D4693) at 37 for 60 minutes under constant agitation. The digested tissues were then filtered through a series of strainers with pore sizes of 100 m, 70 m, and 40 m. Following the tissue dissociation, the resulting cell suspension underwent treatment with red blood cell lysis buffer to eliminate red blood cells. Subsequently, the cell suspension was subjected to centrifugation, and the cell count was determined. The cells were resuspended at a concentration of 1000 cells/l. Due to current technological limitations, achieving a complete digestion of bone tissue to isolate its constituent cells was not feasible. Consequently, single-cell sequencing was conducted to examine cells from the PLL that displayed impending ossification and those from the non-ossified portion. Single-cell libraries were prepared using the Chromium Controller from 10x Genomics (Pleasanton, CA), following the protocol outlined in the Chromium Next GEM Single Cell 3' Reagent Kits (v3.1, 10x Genomics). Libraries were prepared using the Chromium Controller from 10x Genomics. The final library pool was subjected to sequencing on the Illumina Novaseq6000 instrument, generating 150 base-pair paired-end reads. Alignment to the GRCh38 reference genome, quantification of gene expression, and aggregation of sample count matrices were performed using the cellranger pipeline version 6.1.1.