Small RNA from mitochondrial fraction
TRIzol reagent (MRC, Cincinnati, USA) was added to the pellet and L. pyrrhocoris RNA was isolated according to the manufacturer's protocol. To remove DNA contamination, the sample was treated with TURBO DNase (Invitrogen/ Thermo Fisher Scientific, Carlsbad, USA). Thirteen ug of ethanol precipitated mitochondrial-enriched RNA was dephosphorylated using 300 units of Antarctic Phosphatase (New England Biolabs, Ipswich, USA) and purified by TRIzol-chloroform extraction with the Direct-zol RNA miniprep Plus kit (Zymo Research, Irvine, USA). The 5-phosphorylation was performed by the T4 Polynucleotide Kinase (New England Biolabs), followed by another extraction as described above. A final quantity of 2.5 ug of L. pyrrhocoris mitochondrial RNA was commercially sequenced (Macrogen, Seoul, Korea).