Rip immunoprecipitation buffer

RIP assay was carried out in 22RV-1 cells using EZ-Magna RIP^TM RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA) following the manufacturer’s instructions. In briefly, magnetic beads pre-coated with 5 μg normal antibodies against YTHDC1 (Abcam) or rabbit IgG (Millipore) were incubated with cell lysates (1 × 10⁷ cells per sample) at 4 °C overnight. Then, the beads containing immunoprecipitated RNA-protein complex were treated with proteinase K (10 mg/mL) to remove proteins. Then RNAs were purified and the expression of HOXB13 in immunoprecipitated RNAs was assessed by qRT-PCR assay.
To determine the m⁶A levels in HOXB13 mRNA, the m⁶A immunoprecipitation (MeRIP) assay was performed as described previously [49 (link)]. In briefly, 5 μg anti-m⁶A antibody (ABclonal, Wuhan, China) or normal rabbit IgG were bound to magnetic beads (Millipore). A total of 50 μg RNA from 22RV-1 cells was immunoprecipitated using anti-m⁶A antibody in RIP immunoprecipitation buffer (Millipore) overnight at 4 °C. After treating with proteinase K (10 mg/mL), RNA were purified and the expression of HOXB13 in immunoprecipitated RNAs was assessed by qRT-PCR assay. Primers sequences were provided in Supplementary Table S1.

To verify the interaction between NDRG1-OT1 and Argonaute 2 (AGO2), the Magna RNA immunoprecipitation (RIP) Kit (Millipore) was used. MDA-MB-231 breast cancer cells (2 × 10⁷) were harvested with 1% trypsin-EDTA (GIBCO) and lysed with 100 μL RIP lysis buffer containing a proteinase inhibitor cocktail and RNase inhibitor (Millipore). The lysate was centrifuged at 14,000 rpm for 10 min. Then, 900 μL RIP immunoprecipitation buffer (Millipore) containing RIP wash buffer, 0.5% EDTA, and RNase inhibitor were added to the supernatant with 5 μg anti-AGO2 antibodies (cat. no. M00189; Boster Biological Technology, Pleasanton, CA, USA) or 5 μg anti-IgG control antibodies (cat. no. AQ127; Millipore) that were prebound on magnetic beads. The bead mixture was agitated overnight at 4 °C. Ten percent of the supernatant was saved as a record of the input. Beads were washed six times with RIP wash buffer and treated with proteinase K at 55 °C for 30 min. RNA was extracted using NucleoZOL reagent (Machery-Nagel) and reverse-transcribed, and the relative gene expression level was measured by qPCR. The pairs of primers used are listed in Table S3.

T24 cells stably transfected with either the METTL3 overexpress lentiviral or control were UV-irradiated at 254 nm, 400 mJ/cm² (Stratagene Stratalinker), and lysed with RIP lysis buffer (Magna RIP Kit, Millipore, MA) at 4 °C via disruptive sonication. Immunoprecipitations of endogenous DGCR8 were performed using an anti-DGCR8 antibody (1:1000, Abcam, USA) overnight at 4 °C. After washing, the immunoprecipitated protein-RNA complex was analyzed by western blot and treated with Proteinase K. RNAs were extracted by phenol: chloroform: isoamyl alcohol and subjected to qRT-PCR using primers for pri-miRNAs and normalizing to input.
For the m6A RNA binding experiments, the RNAs of T24 cells stably transfected with either the METTL3 overexpress lentiviral or control were isolated and treated with DNase I (Sigma Aldrich, USA). RNAs were fragmented by sonication for 10 s on an ice water mixture. Immunoprecipitations were performed using an anti-m6A antibody (1:1000, Abcam, USA) previously bound to magnetic Dynabeads (Life Technologies, USA) in the RIP Immunoprecipitation buffer (Magna RIP Kit, Millipore, MA) and incubated with DNA-free fragmented RNAs. Beads were then treated with Proteinase K (20 mg/ml) for 1.5 h at 42 °C. RNAs was extracted by phenol: chloroform: isoamyl alcohol and subjected to qRT-PCR using primers for pri-miRNAs and normalizing to input.