Raw RNAseq reads reverse complement to total mitochondrial RNA from yeast N29-dis3A5 derived from BY4741
Sucrose gradient purified mitochondria were treated with cyanase, a highly active and non-specific RNase/DNase, to mitigate cytoplasmic nucleic acid contamination. Mitochondria were re-purified and total RNA was isolated using QIAzol from Qiagen. Total RNA was further purified using the microRNeasy kit from Qiagen, with on column DNase treatment to remove mitochondrial genomic DNA. All RNA-Seq (random sequencing of whole transcriptome) steps were performed by the genomic core facility at the Huntsman Cancer Institute including validation of RNA quality, quantity and purity; library preparation using the Illumina TruSeq Directional RNA protocol with fragmentation to reduce insert size to 30-300 bases, no DSN treatment, no polyA selection, no ribosomal or tRNA subtraction, and no size selection; and 101 cycle directional paired-end sequencing on an Illumina HiSeq2000 platform. Resulting reads were filtered to retain only high-quality reads.