C-to-U RNA Editing in A. kona

C-to-U type RNA editing sites in A. kona mtDNA were predicted using PREPACT (Lenz and Knoop 2013 (link)) (last accessed May 15, 2014). The probability of each candidate editing site was calculated by the percentage of the overlapping predictions against all the references from the output “commons.” Multiple alignment of the gene sequences containing the candidate sites was further checked. Oligonucleotide primer pairs were designed to flank the coding regions of four A. kona mitochondrial genes (nad1, atp6, cob, and cox3) with strong candidate sites (supplementary table S1, Supplementary Material online). For cDNA synthesis, total RNA was extracted and treated with DNase (Thermo). First strand cDNA was synthesized using the Phusion RT-PCR Kit (Thermo) with hexanucleotide random primer mix. PCR amplification of both mitochondrial genomic sequence and cDNA products was performed using the Phusion High-Fidelity DNA Polymerase (Thermo). PCR amplicons were cleaned with ExoSap-IT (GE Healthcare) and sent for direct sequencing.