GSM9474756: KBD2; Homo sapiens; RNA-Seq
Cartilage samples were harvested within an hour post-operation. After washing with phosphate-buffered saline (PBS) containing penicillin and streptomycin, the samples were cut into 1 mm³ fragments and pretreated with 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a 5% CO2 incubator for 30 minutes. The fragments were then centrifuged at 350×g for 5 minutes, and the supernatant was discarded. Subsequent digestion was performed in Dulbecco's Modified Eagle Medium/F12 (Gibco, Grand Island, NY, USA) with 0.2% type II collagenase (Gibco) at 37°C for 10-12 hours. Cell count and viability were determined using a fluorescence cell analyzer (Countstar Rigel S2) with Acridine Orange/Propidium Iodide (AO/PI) staining after erythrocyte removal (Miltenyi 130-094-183). Debris and dead cells were removed following the manufacturer's protocols (Miltenyi 130-109-398/130-090-101). The prepared single-cell suspension was used for cDNA library construction specifically designed for single-cell RNA sequencing using the Chromium Single Cell 3' Library v3 Reagent Kit (10x Genomics, Pleasanton, CA, USA). The pooled cDNA undergoes purification to remove reaction components, followed by PCR amplification to generate sufficient material. Sequencing of this library was performed on the Illumina NovaSeq 6000 platform.