title: GSM5957781: Control_Injured; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell preparation of joint-derived cells were obtained from freshly dissected six weeks post-injury and contralateral knee joints from control and T1DM mice; 4-5 mice per group. Briefly, the tissue around the joint was removed without disrupting the joint or fracture the bone and then washed in PBS to further remove superficial cells. Connective tissue was next severed into digestion media 7.5 mL of 3mg/ml Collagenase type I; (Worthington, Lakewood, NJ) with 100 µg/mL Dnase I (Roche, Basel, Switzerland) in DMEM/F12 for 2 sequential 1h digests at 37°C with shaking at 150 rpm. Following each digest, cells were washed in 5 mL PBS with 1% FBS, supernatants were collected and centrifuged at 500 x g. Red blood cell removal was next performed using ACK lysing buffer (ThermoFisher Scientific, Waltham, MA) followed by immune cell depletion using anti-CD45 magnetic microbeads (Miltenyi Biotec) in combination with LS columns (Miltenyi Biotec).  Cells were resuspended in PBS with 0.04% non-acetylated BSA prior to the single-cell sequencing step using Chromium Single-cell 3ʹ GEM, Library & Gel Bead Kit v3 (10× Genomics) on a 10x Genomics Chromium Controller following manufacturers protocol. Single-cell 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.