2.2 DNA extraction, sexing and sequencing
High molecular weight DNA was extracted from flash frozen heart and kidney using the Nanobind Tissue Big DNA Kit v1.0 11/19 (Circulomics). A Qubit fluorometer was used to quantify DNA concentrations with the Qubit dsDNA BR assay kit (Thermo Fisher Scientific). RNA was extracted from heart, spleen, kidney, gonads, brain, and liver stored in RNA later using the RNeasy Plus mini Kit (Qiagen) with RNAse-free DNAse (Qiagen) digestion. RNA quality was assessed via Nanodrop (Thermo Fisher Scientific). We extracted DNA for population genomics from blood and feather samples using the Monarch® Genomic DNA Purification Kit (New England BioLabs, Victoria, Australia). We quantified DNA concentrations using a Qubit 3.0 fluorometer (yield range 10.3 – 209 ng μl-1, Table S1) and standardised the concentration of each sample to 10-30 ng µl-1 DNA for 20-25 μl and determined the sex of individuals using a polymerase chain reaction (PCR) protocol adapted from Fridolfsson and Ellegren (1999, Supplementary file S1). We arranged the samples on a single 96 well plate, containing five technical replicates of the samples with the highest DNA concentrations, an additional 21 non-technical replicates including all of the King Island samples, five extra samples from mainland Tasmania and one negative control.
Double-digest restriction associated DNA (ddRAD) sequencing following Peterson et al. (2012) was undertaken at the Australian Genome Research Facility, Melbourne on an Illumina NovaSeq 6000 platform using 150bp paired-end reads. Samples were first quantified using Quantifluor and visualised on 1 % agarose e-gel to ensure all samples exceeded the minimum input DNA quantity of 50 ng. Three establishment samples with at least 250 ng DNA that were representative of the distribution of the samples (2 Tasmanian scrubtits, 1 King Island scrubtit) were used to determine the optimal combination of restriction enzymes, which wereEcoRI and HpyCH4IV . Further details on the library preparation protocol are provided in Supplementary file S1.