GSM5258024: Uninjured; Mus musculus; RNA-Seq
Mice were euthanized and injured and uninjured hindlimbs were collected by removing the legs 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). Articular cartilage from tibia and femora was isolated by cutting ~1 mm of tissue from the end of both long bones at the knee joint. For each experimental group, cartilage tissue from 5 mice was pooled, and digested to a single cell suspension in 5 mL of 0.2% Collagenase 2 solution (2mg/mL Thermo Fisher Scientific, Waltham, MA, USA) while shaking at 37 °C for a total of 2 hours in 30-minute intervals. Interval fractions were filtered through a 70 μm Nylon cell strainer into DMEM/F12 with 10% fetal bovine serum (FBS) and kept on ice. Remaining undigested cartilage tissue was further digested in 5 mL of fresh Collagenase 2 digestion media. After the final digestion interval, cells were pelleted via centrifugation for 10 min at 500 G at 4oC, incubated on ice with ACK lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA), for red blood cell removal. Cells were stained with the following antibodies for flow cytometry and FACS analysis: CD45 APC-Cy7 (BioLegend, San Diego, CA, USA, 103116 (1:100)), Ter119 APC (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-102-290, (1:10)), and DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Immune and Erythroid contamination was depleted via double negative selection (CD45-; Ter119-) using the BD FACSMelody (San Jose, 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 targeting approximately 50,000 reads per cells.