mt-genome of lasiorhinus krefftii: tissue
We constructed double stranded libraries from 20 uL of DNA extract with truncated Illumina adapters containing dual 5-mer barcodes. The blunt end repair, adapter ligation and Bst fill-in were performed following the protocol of Meyer and Kircher (2010) with MinElute (Qiagen) clean-ups between each reaction step. Following library preparation, adapter-ligated DNA was amplified in eight separate 25 L reactions containing 1 High Fidelity Buffer (ThermoFisher Scientific), 2 mM MgSO4, 250 M each dNTP, 500 nM IS7_short_amp.P5 (Meyer and Kircher 2010), 500 nM IS8_short_amp.P7 (Meyer and Kircher 2010), and 1 U of Platinum Taq DNA Polymerase, High Fidelity (ThermoFisher Scientific). Thermocycling conditions were: 94C for 2 min, 13 cycles of 94C for 15 s, 60C for 15 s, 68C for 30 s, followed by 68C for 10 min. All eight reactions for each library were pooled and then purified using AmpureXP beads at a ratio of 1.8 as per manufacturer's instructions.  We assessed the relative DNA yield of each library preparation protocol using a Qubit fluorometer (Thermo Fisher Scientific) and the dsDNA High Sensitivity Assay Kit. Genomic libraries were enriched for wombat mtDNA using hybridisation capture with in-house synthesised wombat mtDNA RNA baits as described by Richards et al. (2019). In short, we amplified the mtDNA genome (excluding the control region) in three, overlapping long-range PCRs from a single modern NHNW DNA extract and then produced biotinylated RNA probes via in vitro transcription, fragmentation and biotinylation. Genomic DNA libraries were hybridised to the RNA probes following the MyBaits User Manual Version 3.02 (http://www.arborbiosci.com/wp-content/uploads/2017/10/MYbaits-manual-v3.pdf).