Normal rabbit igg

The ChIP assay was performed using the BeyoChIP™ ChIP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, 1 × 10⁷ CRC cells were prepared and then cross-linked with 1% formaldehyde for 15 min at room temperature. The cross-linking process was quenched by adding 0.125 M glycine. Chromatin was isolated with the lysis buffer provided in the kit. Afterwards, sonication was performed to shear the DNA to 200–1000 bp. Immunoprecipitation of cross-linked protein/DNA was performed by incubating anti-YBX1 antibody (Proteintech, IL, USA) or normal rabbit IgG (Proteintech, IL, USA) with sheared chromatin overnight at 4 °C followed by incubation with Protein A/G Magnetic Beads for 2 h. Subsequently, cross-links of protein/DNA were released with 5 M NaCl, and the DNA was purified using a PCR Clean Up Kit (Beyotime, Shanghai, China). Finally, the purified DNA was subjected to qRT–PCR using the P1-P9 primer sequences listed in Additional file 1: Table S4.

For immunoprecipitation-sequencing, total RNA was isolated from peritoneal macrophage. Briefly, macrophages with and without Ang II treatment were re-suspended and lysed in RIP lysis buffer, and the lysates were used as input. Then, lysates were incubated with ALKBH5 antibody in RIP immunoprecipitation buffer at 4 °C for 4 hours. Normal Rabbit IgG (Proteintech) was used as a negative control. The lysates were immunoprecipitated by Protein A/G beads (Repligen) at 4 °C for an additional 2 hours. The magnetic bead-bound complexes were centrifuged and washed for 5 times. The precipitated RNA samples were extracted and purified with phenol, chloroform and isoamyl alcohol, and the immunoprecipitated RNA was used to construct RNA libraries. Then, library sequencing was performed by illumine HiSeq 4000 sequencer with 150 bp paired-end reads. Data analysis was performed according to the published procedure.