GSM6044906: D1; Mus musculus; RNA-Seq
Mice were euthanized and femur and tibia of injured and uninjured hindlimbs were dissected, and superficial tissue were thoroughly removed, taking care to maintain the integrity of the knee joint. Joint residing cells were liberated by first dissociating the two bones into 7.5 mL of 3% Collagenase 1 solution (Worthington Biochemical, Lakewood, NJ; CLS-1) and 100 µg/mL DNase I (Roche, Basel, Switzerland; 11284932001) in DMEM/F12 shaking at 37 °C for two 1-hour digests followed by PBS wash steps between digests. Cells released from the joint were filtered through a 70 μm nylon cell strainer prior to red blood cell (RBC) lysis using ammonium-chloride-potassium (ACK) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA; A1049201). Separation of immune cells was performed using CD45 conjugated magnetic microbeads followed by Miltenyi Biotech MACS separation with LC columns for CD45+ cell enrichment. Both immune (CD45+) and non-immune (CD45-) cell populations were independently sequenced using a Chromium Single Cell 3' Reagent Kit and Chromium instrument (10X Genomics, Pleasanton, CA). Library preparation was performed according to manufacturer's protocol.