Paleogenetic Sample Preparation and Extraction

Samples were prepared and extracted in the paleogenetics clean room at the Institute for Archaeological Sciences, University of Tübingen (INA). The surface of the dedicated sampling hood was cleaned with HPLC water and UV irradiated by an internal light source between uses. Any calculus was removed from the surfaces of the teeth using dental scalers that had been rinsed with bleach and HPLC water and UV irradiated for 10 minutes between uses. Large calculus samples were pulverized with a tube pestle. Teeth were then sectioned horizontally at the cementoenamel junction and dentin was drilled from the pulp chamber using a dental drill. For calculus samples weighing over 20 mg, half the pulverized material was carried over for extraction. For dentin samples over 70 mg, aliquots of approximately 50 mg were taken for extraction. Dentin and calculus samples were extracted using a previously described silica-based method^{75 (link)}. In brief, samples were submerged in a digestion buffer with final concentrations of 0.45 M EDTA and 0.25 mg/mL proteinase K (Qiagen, the Netherlands) and rotated at 37 °C until decalcified. After incubation, samples were centrifuged and the supernatant was purified using a 5 M guanidine-hydrochloride binding buffer with High Pure Viral Nucleic Acid Large Volume kits (Roche, Switzerland). The extracts were eluted in 100 μl of a 10 mM tris-hydrochloride, 1 mM EDTA (pH 8.0), and 0.005% tween-20 buffer (TET). One extraction blank was prepared for every ten samples, and one positive control of cave bear bone powder was processed alongside each extraction batch to ensure efficiency. The extracts were quantified using a Qubit High Sensitivity fluorometer (Life Technologies) (Supplementary Table S1).