GSM8834080: wt-control-3; Mus musculus; RNA-Seq
Total RNA was isolated from liver tissue using TRIzol reagent (Invitrogen) according to the manufacturer's protocol with minor modifications. Briefly, approximately 50–100 mg of liver tissue was homogenized in 1 mL of TRIzol reagent using a Polytron homogenizer. The homogenate was incubated at room temperature for 5 minutes to ensure complete dissociation of nucleoprotein complexes. Subsequently, 0.2 mL of chloroform was added per 1 mL of TRIzol, and the mixture was vigorously shaken for 15 seconds, followed by incubation at room temperature for 3 minutes. Samples were centrifuged at 12,000 × g for 15 minutes at 4°C to separate the phases. The aqueous phase containing RNA was carefully transferred to a new RNase-free tube, and RNA was precipitated by adding 0.5 mL of isopropanol per 1 mL of TRIzol used. After incubation at room temperature for 10 minutes, samples were centrifuged at 12,000 × g for 10 minutes at 4°C to pellet the RNA. The RNA pellet was washed once with 1 mL of 75% ethanol (prepared with DEPC-treated water), followed by centrifugation at 7,500 × g for 5 minutes at 4°C. The supernatant was discarded, and the RNA pellet was air-dried for 5–10 minutes. The pellet was resuspended in 20–50 µL of DEPC-treated water and incubated at 55–60°C for 10 minutes to ensure complete dissolution. To remove any contaminating genomic DNA, RNA samples were treated with DNase I (RNase-free) according to the manufacturer's instructions. Briefly, 1 µg of RNA was incubated with 1 U of DNase I in the presence of 1× reaction buffer and 1 U/µL RNase inhibitor at 37°C for 30 minutes. The reaction was terminated by adding EDTA to a final concentration of 2.5 mM and incubating at 65°C for 10 minutes. RNA concentration and purity were determined using a spectrophotometer (NanoDrop), with an A260/A280 ratio of ~2.0 indicating high-purity RNA. RNA integrity was verified by agarose gel electrophoresis or using a Bioanalyzer. RNA samples were stored at -80°C until further use. The Illumina Truseq kit was used to generate libraries.
GEO
GSM8834080_r1