m5C-RIP Analysis of RNA Modifications

Total RNA was extracted from HepG2.2.15 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). m⁵C-RIP was performed as previously described [55 (link)]. Briefly, 300 μg of RNA were resuspend in immunoprecipitation (IP) buffer (150 mmol/L NaCl, 0.1% NP-40, 10 mmol/L Tris-HCl pH 7.4) and incubated with an anti-m⁵C antibody (ab10805, Abcam) or normal rabbit/mouse IgG antibody (Proteintech) overnight at 4 °C. The RNA and antibody mixture was then incubated with 35 μL of magnetic beads (New England Biolabs) for 2 h at 4 °C. Beads were washed with IP buffer six times and then incubated with 300 μL of elution buffer (5 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA pH 8.0, 0.05% SDS, 4.2 μL 20 mg/mL proteinase K) at 50 °C for 1.5 h. Eluted RNA was purified using phenol/chloroform. Immunoprecipitated RNA was used for cDNA synthesis using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme) according to the manufacturer’s protocol. The relative RNA level was measured by quantitative PCR (qPCR) using Hieff® qPCR SYBR® Green Master Mix (Yeasen Biotech Co., Shanghai, China) on a CFX Connect real-time system (Bio-Rad Laboratories, Hercules, CA, USA). The primers used for RT-qPCR were listed in Table S6. At least three samples in each qPCR analysis were prepared, and three independent experiments were performed.