Genomics chromium controller instrument and chromium single cell 3 v3 reagent kits

The scRNA‐Seq libraries were generated using the 10X Genomics Chromium Controller Instrument and Chromium Single Cell 3′ V3 Reagent Kits (10X Genomics, Pleasanton, CA). Briefly, cells were concentrated to 1000 cells per µL and ≈8000 cells were loaded into each channel to generate single‐cell gel bead‐in‐emulsions (GEMs), which results in expected mRNA barcoding of 6500 single‐cells for each sample. After the RT step, GEMs were broken and barcoded‐cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A‐tailed, ligated with adaptors and index PCR amplified. The final libraries were quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific) and the size distribution of the libraries were determined using a High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced using an Illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired‐end run.

sc-RNA-seq data, GSE104782 and GSE169454 (26 (link), 27 (link)), were obtained from the Gene Expression Omnibus of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/geo/). The GSE104782 dataset contains 1600 chondrocytes of 10 patients with OA undergoing knee arthroplasty surgery, and cells are labeled with OA grades according to the OARSI grading system (grade 0; n = 320, grade 1; n = 320, grade 2; n = 320, grade 3; n = 320, grade 4; n = 320). These scRNA-seq libraries were generated using a Kapa Hyper Prep Kit (Kapa Biosystems, Wilmington, MA, USA) and sequenced using the Illumina HiSeq 4,000 platform (Novogene, Beijing, China) with a read length of 150 bp. The GSE169454 dataset contains 7 scRNA-seq dates. These scRNA-Seq libraries were generated using the 10X Genomics Chromium Controller Instrument and Chromium Single Cell 3’ V3 Reagent Kits (10X Genomics, Pleasanton, CA, USA) and sequenced using the Illumina HiSeq 4,000 platform (Novogene) with a read length of 150 bp.