title: GSM2692128: LRPPRC_KO_RNase_Untreated; Mus musculus; RNA-Seq
library_construction_protocol: Mitochondria were isolated from homogenized hearts and isolated by differential centrifugation as described previously (Lagouge et al., PLoS Genetics, 2015; Mourier et al., Human Molecular Genetics, 2014), with some modifications. Hearts or livers were cut and washed three times with ice cold PBS, and once with mitochondrial isolation buffer (MIB) containing 310 mM sucrose, 10 mM Tris-HCl and 0.05 % BSA (w/v) by centrifugation at 4,500 g for 1 min at 4°C. Heart pieces were homogenized in 5 ml of fresh MIB using a Potter S pestle (Sartorius). The homogenate was centrifuged at 1,000 g for 10 min at 4˚C and the supernatant was centrifuged at 4,500 g for 15 min at 4°C to isolate mitochondria. Crude mitochondrial pellets were suspended in MIB supplemented with 1x Complete EDTA-free protease inhibitor cocktail (Roche). Protein concentration was determined by the Bradford or BCA method using BSA as a standard. Mitochondria (2 mg.ml-1) were lysed by addition of 200 µl of lysis buffer (100mM Tris-HCl, 100mM NaCl, 40mM MnCl2, 2mM dithiothreitol pH 7.5, 0.1% TritonX-100). The concentration of the RNase A (10 U/µl), RNase T1 (0.1 U/µl) or RNase If (0.01 U/µl) were optimized and added to each mitochondrial lysate or purified RNA to generate 15-55 nt size fragments. All incubations were carried out at 37˚C for up to 30 minutes and reactions were ended by addition of 700 µl Qiazol, followed by RNA isolation using the miRNeasy Mini Kit (Qiagen). We used mitochondria lysed and mock treated as controls for the footprinting assay. The libraries were constructed using the Illumina TruSeq Small RNA Sample Prep Kit and deep sequencing of the mitochondrial small RNAs was performed by Australian Genomic Research Facility (Melbourne, Australia) on an Illumina GAII (Illumina, CA) according to the manufacturer’s instructions with one modification, sample isolation from the PAGE gel after adaptor ligation was performed with a modified set of size markers to facilitate capture of small RNAs between 15-55 nt.