GSM7870479: mitoDMS KO2; Homo sapiens; OTHER
RNA was extracted following the Trizol manufacturer's specifications. The aqueous phase was transferred to a new tube, and an equal volume of 100% isopropanol and 3 μL of glycogen were added to precipitate the RNA. The samples were incubated at -80 oC overnight and centrifuged at 15,000 xg for 45 min, at 4 °C. RNA was resuspended in 50 μL of RNAse-free water and quantified by measuring absorbance at a wavelength of 260 nm with Nanodrop (ThermoFisher Scientific). Sequencing libraries were generated using the xGen Broad-range RNA library prep kit from IDT, following the manufacturer's specifications, with the following modifications. The RNA fragmentation step was conducted by combining 1 μg of RNA with 1 μL of reagent F1, 4 μL of F3, and 2 μL of F4, and incubating at 95 °C for 2 min. The samples were immediately placed on ice and combined with a mixture of 1 μL 10 mM TGIRTIII reverse transcriptase (RT), 1 μL R1 reagent, and 1 μL 0.1 M DTT. This RNA:RT mixture was incubated for 30 min at room temperature before adding 2 μL of reagent F2, and then placed in a thermocycler for 10 min at 20°C, 10 min at 42 °C, and 60 min at 55 °C. The thermocycler was programmed to pause at 55°C, at which point 1 μL of 4M NaOH was added and programmed to continue at 95°C for 3 minutes. At this point, the sample was removed from the thermocycler and placed on ice, and 1 μL of 4M HCl and 27 μL of low EDTA TE were added. After the indexing PCR, the indexed libraries were resolved on an 8% TBE gel for 55 min at 180 V and size-selected for products ~300 nt. The products were sequenced with NovaSeq6000 system (paired-end run,150 cycles).