Protein g coupled magnetic dynabeads

For RIP assay, cells were harvested and lysed in polysome lysis buffer [100 mM KCl, 10 mM HEPES (pH 7.0), 0.5% NP40, 5 mM MgCl₂, 1 mM DTT, 80 U/ml RNase inhibitors, and protease inhibitor cocktail] for 10 min on ice. Cell lysates were sonicated to fragment chromatins and RNAs and centrifuged. The protein concentration in the supernatant was measured with the BCA protein assay kit (Thermo Fisher Scientific, USA). Proteins were incubated with Protein G-coupled Magnetic Dynabeads (Life Technologies, USA) pre-coated with rabbit anti-METTL14 antibody, anti-FOXO1 antibody, anti-m6A antibody, or IgG control overnight at 4°C. Prior to the incubation, 1/10 of the supernatant was set aside to be used as input. After incubation, samples were washed 5 times with NT buffer [50 mM, Tris-HCl (pH 7.4), 1 mM MgCl₂, 150 mM NaCl, and 1% Triton X-100]. Immunoprecipitated RNAs and input RNAs were extracted using TRIzol reagent (Invitrogen). For meRIP-seq analysis, reads uniquely mapped to the genome were subjected to calling the peaks in the enriched regions with a fold enrichment of at least 2 over input reads.

Scr and miR-375 transfected cell (∼3.5×10⁶) were scraped of culture flasks on ice in gentle lysis buffer (20 mM TRIS pH 7.5, 10 mM NaCl, 0.5% NP-40, 2 mM EDTA supplemented with RNase inhibitor RNaseOut (Invitrogen) and Complete Mini Protease Inhibitor Cocktail (Roche)) and hypertonically lysed by increasing the NaCl concentration to 150 mM. After centrifugation at 4°C and 19,000*g for 10 minutes the supernatant was collected and subjected to immunoprecipitation (10% was used for input control) by incubation with monoclonal Ago2 antibody (11A9) (Sigma-Aldrich) -bound Protein G-coupled Magnetic Dynabeads (Life technologies) (15 mg 11A9 per 25 ml beads) following the manufacturer's recommendation. Anti-FLAG immunoprecipitation was done in parallel as a negative control (antibody F1804, Sigma). The beads were washed 5 times in ice cold washing buffer (50 mM TRIS pH 7.5, 150 mM NaCl and 0.05% NP-40). Total RNA from input, Ago2-IP and FLAG-IP complexes was purified using QIAZol (Qiagen).

The SW620.625 and control cells were scraped off culture flasks on ice in gentle lysis buffer (20 mM TRIS pH 7.5, 10 mM NaCl, 0.5% NP-40, 2 mM EDTA supplemented with RNase inhibitor RNaseOut (Life Technologies) and Complete Mini protease inhibitor cocktail (Roche Applied Science)), incubated for 5 min before being hypertonically lysed by increasing the NaCl concentration to 150 mM and incubated for additionally 5 min on ice. After 4 °C centrifugation at 19,000g for 10 min, the supernatant was collected and subjected to pull-down (10% was used for input control) by incubation with monoclonal AGO2 antibody 11A9 (Sigma-Aldrich, Cat. #SAB4200085)-bound Protein G-coupled Magnetic Dynabeads (Life Technologies; 15 mg 11A9 per 25 μl beads) following the manufacturer's recommendation. All washing steps were performed in ice cold washing buffer (50 mM TRIS pH 7.5, 150 mM NaCl and 0.05% NP-40), and total RNA from input and immunoprecipitate fractions purified with QIAzol (Qiagen). A parallel pull-down using monoclonal M2 anti-FLAG antibody (Sigma-Aldrich, Cat. #F1804) was performed as negative control.