title: Mitogenomics of the Extinct Cave Lion, Panthera spelaea (Goldfuss, 1810), Resolve its Position within the Panthera Cats
design: Samples of cortical bone were taken (approx. 1 cm3) using a Dremel powertool and reduced to powder in a Mikrodismembrator. DNA extraction was performed as described by Orlando et al. (2013) in a dedicated ancient DNA laboratory at the Centre for GeoGenetics in Copenhagen, in parallel with negative extraction controls.   The DNA extract and negative control were then built into genomic libraries using the NEB E6070 kit (New England Biolabs), following a protocol slightly modified from that by Vilstrup et al. (2013). Briefly, extract (30 µL) was end-repaired and then passed through a MinElute column (Qiagen). The collected flow-through was then adapter-ligated and passed through a QiaQuick column (Qiagen). Adapter fill-in reaction was then performed on the flowthrough, before final incubation at 37°C (30 min) followed by inactivation overnight at -20°C.   We then amplified the DNA in a 50 µL reaction, using 25 µL of library for 12 cycles under the following reaction conditions. Final concentrations were 1.25 U AccuPrime™ Pfx DNA Polymerase (Invitrogen), 1× AccuPrime™ Pfx reaction mix (Invitrogen), 0.4 mg/mL BSA, 120 nM primer in PE, and 120 nM of a multiplexing indexing primer containing a unique 6-nucleotide index code (Illumina).   PCR cycling conditions consisted of an initial denaturation step at 95°C for 2 min, followed by 12 cycles of 95°C denaturation for 15 s, 60°C annealing for 30 s, and 68°C extension for 30 s. A final extension step at 68°C for 7 min was also included. Amplified libraries were first checked for presence of DNA on a 2% agarose gel before purification using the QIAquick column system (Qiagen) and quantification on an Agilent 2100 BioAnalyzer. Quantified libraries were communally pooled in equimolar ratios and sequenced as single-end reads (100 bp) on an Illumina HiSeq2000 platform at the Danish National High-Throughput Sequencing Centre.