GSM8111686: Zhx5-2, liver, WT+CCL4; Mus musculus; OTHER
Trial procedures were performed with minor modifications, as previously reported.10 Liver tissue samples, each weighing 200 mg, were immediately frozen in liquid nitrogen and physically disrupted in 1.5 mL of lysis buffer. The buffer composition included 1% NP-40, 200 mM KOAc, 25 mM HEPES-KOH pH 7.2, 10 mM MgCI2, and 4 mM CaCI2. Following a 5-min incubation of the liver tissue on ice in the lysis buffer, the lysate was centrifuged at 9000 rpm for 5 min at 4°C. The resulting supernatant was separated into two 200 μL aliquots to generate an input sample and a Ribo-bound sample.  The sample was then diluted with ddH2O at a 1:1 ratio, and MNase was added at a final concentration of 0.2 U/μL lysate, followed by a incubation at 37°C for 30 min. RNA extraction was carried out using Trizol. The RNA was resuspended in a 10 μL PNK solution (1 μL 10X buffer A, 1 μL PNK, 1 μL ATP, and RNA in 7 μL H2O), with a subsequent 30-min incubation at 37°C. A total of 10 μL of RNA loading dye was added to the mixture. Gel electrophoresis was performed at 180 V for 75 min. The gel was immersed in 25 mL 1X TBE with a 1:10000 SybrGold solution for 5 min, exposed under UV light, printed at actual size, and placed in a tube.  Subsequently, 400 μL of 400 mM NaOAc was added, and the gel slice was frozen at -80°C for 10 min and thawed at 95°C for 5 min. The gel slice was then thoroughly homogenized using a pestle or pipet tip and subjected to RNA extraction through three cycles of heating (95°C, 5 min, 20 min of shaking). The extracted gel slice was loaded onto a SpinX column, centrifuged for 10 min at maximum speed, treated with 1 mL ethanol and 1 μL Glycogen, and incubated for 1 h at -20°C. After centrifugation for 15 min at 4°C and washing with cold 80% ethanol, the pellet was dried for 10 min before being resuspended in 7 μL of water. NEB Next Small RNA Library Prep Set for Illumina was employed for library construction.