Sterile Cartilage Extraction and Microbiome DNA Isolation

Knee joints were dissected in a biosafety cabinet using sterilized, UV- and DNA/RNA-decontaminated (DNA-Zap solution, ThermoFisher, Waltham, MA, USA) instruments following skin and synovial capsule sterilization with chlorhexidine. Full-thickness articular cartilage was removed from the tibia and femur using a disposable, sterile #11 blade and immediately flash frozen and stored in liquid nitrogen. Later, cartilage samples were cryogenically ground using a Precellys Cryolys instrument (Bertin, Bretonneux, France) at 0 °C and DNA isolated using a DNEasy kit (Qiagen). Cecal contents were flash frozen in liquid nitrogen then DNA extracted using a Qiagen QIAamp DNA microbiome kit. All plasticware and reagents were decontaminated by a 30-min UV exposure as previously described [8 , 12 (link), 13 (link)]. PCR master mixes and tubes were further enzymatically decontaminated with dsDNAse (PCR decontamination kit, Arcticzymes, Tromsø, Norway).