NextSeq 550 sequencing; A landscape of mouse mitochondrial small non-coding RNAs
A landscape of mouse mitochondrial small non-coding RNAs
A miRNA analysis on Mitochondria of 4 tissues (liver, gastrocnemius skeletal muscle, testis, white adipose tissue) was performed by RNA-seq in 3 wild-type male CD1 mice, ~3 months old, maintained in a temperature-controlled room at 22 ± 2°C under a 12 h dark/light cycle with a standard pellet diet and unlimited access to water. Mitochondria were purified from freshly excised tissues (liver, gastrocnemius skeletal muscle, testis, white adipose tissue) using Qproteome Mitochondrial Isolation kit (Qiagen) according to manufacturer's instructions with minor modifications and performing all the steps at 4°C. In order to remove RNA of nuclear origin present in the cytosol and eventually adsorbed on the outer mitochondrial membrane, mitochondria were treated with RNase A solution. Total RNA was purified from mitochondria by miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. The RNA integrity and concentration was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies). The mitochondrial RNA enrichment and eventual cytosolic RNA contamination were checked by RT-qPCR. Mitochondria and cytosolic RNAs were retrotranscribed by SensiFAST cDNA Synthesis kit (Bioline). Indexed libraries were prepared with NEXSmallRNA Seq v3 (PerkinElmer) according to the manufacturer's instructions. Libraries were quantified using the Tape Station 2200 (Agilent Technologies) and Qubit Fluorometer 1.0 (Invitrogen Co., Carlsbad, CA). Libraries were pooled such that each index-tagged sample was present in equimolar amounts, with final concentration of the pooled samples of 2nM.