*4.5. Bulk RNA Sequencing and Data Analysis*

The knee joint RNA from 10-week-old, 62-week-old, and 95-week-old mice was isolated as described before [10]. Briefly, injured and contralateral joints (*n* = 3–6 per group) were dissected, chopped, and homogenized in Qiazol (79306, Qiagen, Valencia, CA, USA). Total RNA was purified using RNeasy Mini Kit (QIAGEN Inc., Germantown, MD, USA) according to the manufacturer's protocol and the RNA integrity was assessed using a bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Poly(A)+-enriched cDNA libraries were generated using the Illumina TruSeq RNA Library Prep kit v2 (Illumina Inc., Hayward, CA, USA). The sequencing was performed using an Illumina (Illumina Inc., Hayward, CA, USA) NextSeq 500 instrument to generate 75 bp single-end reads. The quality of sequencing data was checked using FastQC (version 0.11.5) software [http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc]. Sequence reads were mapped to the mouse reference genome (mm10) using STAR (version 2.6) [39]. A matrix of raw counts per gene was generated using "featureCounts" from Rsubread package (version 1.30.5) [40]. RUVseq was used to determine factors of unwanted variation [41]. Differentially expressed genes were identified using edgeR, adjusting for factors of unwanted variation [42]. Genes with fold changes >1.5 and false discovery rate (FDR) adjusted *p*-value less than 0.05 were considered as significantly differentially expressed. Heatmaps were generated using heatmap.2 function in R package 'gplots'.

#### *4.6. Single Cell RNA-Seq (scRN-Aseq) and Data Analysis*

Ten-week-old C57Bl/6J mice were euthanized, and right and left hindlimbs were collected by removing the leg at the hip joint and storing on ice in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12) (Thermo Fisher Scientific, Waltham, MA, USA). The articular cartilage of the knee joint was isolated from the femur and tibia by cutting 0.5–1 mm of tissue from the end of both long bones. Cartilage tissue was digested to a single cell suspension in 1 mL of 0.2% Collagenase 2 solution (Thermo Fisher Scientific, Waltham, MA, USA) while shaking at 37 ◦C for 2 h. Fractions were collected at 30 min intervals, filtered through a 70 μm Nylon cell strainer, and then resuspended in DMEM/F12 with 10% fetal bovine serum (FBS). Remaining undigested cartilage tissue was further incubated in 1 mL of fresh Collagenase 2 digestion media.

Following digestion, cell suspensions were pelleted via centrifugation for 10 min at 500 G, then resuspended in ACK lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA), and incubated for 10 min on ice in order to remove red blood cells. Cell suspensions were next resuspended in 100 uL of PBS + 10% FBS. CD45+ and Ter119+ cell depletion was accomplished using CD45 and Ter119 MACSmicrobeads (Miltenti Biotec, Sunnyvale, CA, USA) and running cell samples on an LS MACScolumns (Miltenti Biotec, Sunnyvale, CA, USA) following the manufacturer's instructions. Final cell counts were performed using a hemocytometer and cells were resuspended in PBS + 0.04% nonacetylated BSA before introduction into a Chromium Controller (10x Genomics, Pleasanton, CA,

USA). Library preparation was performed using Chromium Single Cell 3 GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA; Catalog no. 1000075) following the manufacturer's protocol and sequenced using Illumina NextSeq 500. Sequencing data were demultiplexed, quality controlled, and aligned to the mouse genome (mm10) using Cell Ranger (10x Genomics, Pleasanton, CA, USA). Data analysis was performed using Seurat (version 3.1.1) [43].
