title: GSM8823955: mitoDMS FASTKD5 knockout 2; Homo sapiens; OTHER
library_construction_protocol: 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 for 1 hour and centrifuged at 15000 g for 20 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 (Thermo Fisher Scientific). 10 μg of RNA was DNase-treated using the TURBO DNase kit (Thermo Fisher Scientific). After treatment, the RNA was purified using RNA Clean and Concentrator −5 kit (Zymo Research) according to the manufacturer's protocol. DNase treatment and column purification were repeated two additional times to ensure the complete removal of all DNA. Sequencing libraries were generated using the xGen Broad-range RNA library preparation kit from IDT, according to the manufacturer's instructions, 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 Induro Reverse Transcriptase (NEB), 1 μl R1 reagent, and 1 μl 0.1 M DTT. This 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. Then, 1 μl of 4M NaOH was added, and the samples were incubated 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 IDTE (10 mM Tris, 0.1 mM EDTA) 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 using the NovaSeq X Plus platform (Illumina) with PE 150, 250M reads per sample.