Anti ctbp2

The CLIP-Seq Kit (BersinBio) was used according to the manufacturer’s protocol. Before cross-linking, 4-thiouridine (Macklin, 13957-31-8) was supplemented to the cell culture medium at a final concentration of 100 μM to facilitate the cross-linking of RNA and proteins. Following a wash with prechilled 1× PBS, the cells were exposed to 365 nm UV light for 10 minutes at a dosage of 0.15 J/cm². Subsequently, the cells were meticulously harvested and subjected to centrifugation for supernatant removal. The lysed cells were then subjected to digestion using a cell lysis buffer and RNase T1, and 100 μL of the resulting supernatant was collected as the input sample. The remaining samples were incubated overnight with either anti-CTBP2 (Cell Signaling Technology, 13256S), anti-PCIF1 (Abcam, ab271081), or IgG antibodies. Immunoprecipitation (IP) was performed using Protein A/G magnetic beads, followed by sequential washing steps. DNase I digestion and bead washing were subsequently carried out, after which protein digestion was performed, and the supernatant from the immunoprecipitated samples was collected. Finally, both the input and immunoprecipitated samples’ supernatants were subjected to protein digestion to prepare the RNA samples for subsequent high-throughput sequencing analysis.