GSM7029293: MitoRP_K562 2; Homo sapiens; OTHER
Upon removing media, cells were rinsed with ice-cold PBS and resuspended in mitoribosome lysis buffer (0.25% lauryl maltoside,50 mM NH4Cl, 20 mM MgCl2, 0.5 mM DTT, 10 mM Tris, pH 7.5, and 1× EDTA-free protease inhibitor cocktail. Mouse lysate was spiked in to a final volume of 5% and lysates were RNAse digested for 30 min at room temperature to generate mitoribosome footprints using 8 units/µL of RNase If (NEB). The digestion was then inhibited by the addition of 80 units SUPERaseIn (Thermo Fisher Scientific) followed by centrifugation at 10,000 RPM for 5 min. The mitochondrial monosome fraction was collected after sucrose density gradient fractionation and RNA fragments 28 - 40 nt were gel purified. Custom library prep using 3' linker /5rApp/NNNNNNNNNNCTGTAGGCACCATCAAT/3ddC/, reverse transcription with primer /5Phos/NNNNGATCGTCGGACTGTAGAACTCTGAACCTGTC/iSp18/CACTCA/iSp18/CAAGCAGAAGACGGCATACGAGATATTGATGGTGCCTACAG, and circularization before PCR with Illumina primers  513 /5rApp/NNNNNNCTGTAGGCACCATCAAT/3ddC/) and 5' linker