RNA sequencing of PET127 mutants overexpressed using the galactose inducible promoter

In Saccharomyces cerevisiae, PET127 is a mitochondrial RNA exonuclease known to interact with the mitochondrial RNA polymerase RPO41. The nature of the interaction between PET127 and RPO41 is unclear. Here, we overexpressed different PET127 mutants for five hours utilizing the galactose inducible promoter on high-copy plasmids, collected samples, and performed RNA sequencing to understand the mitochondrial RNA landscape in each condition. The mutants tested were PET127 lacking the nuclease activity (pet127-nd) and two truncated forms of PET127: either lacking the RPO41 binding region (pet127∆(48-215)), or only containing the RPO41 binding region (pet127(1-215)).

Methods
RNA was isolated from 4.4 x 107 cells of cells using acid phenol and purified using RNeasy kit (Qiagen) with on column DNAse treatment (Qiagen). Isolated total yeast RNA were processed using Ribozero rRNA removal for Yeast (Illumina). Samples were collected upon addition of galactose and 5 hours after the addition of galactose. Sequencing was performed on HiSeq2000 with 40 bases single end run with 8 + 8 nucleotide indexes.