mRNA Methylation Complex Immunoprecipitation

Each sample used 100 µl Protein-G DYNA beads blocked over night at 4 °C in 500 µl of IP lysis buffer (50 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Triton X-100, 10% Glycerol, 1 mM DTT) containing 1% BSA and 2–10 µg of antibody. Beads were subsequently washed 3 times in 1 ml IP lysis buffer. Cells were washed with PBS before lysis in IP lysis buffer containing protease inhibitor, DNase 1, RNase A and DTT. Extracts were cleared by centrifugation at 13 200 rpm, 5 minutes at 4 °C. Cell extracts were incubated with antibody bound beads for 2 hours at 4 °C. Beads were washed 3 times in 1 ml ice cold IP lysis buffer. After the final wash beads were resuspend in 72 µl 1 M Arginine-HCl pH 3.5, incubated for 1 minute on ice and spun for 1 minute at 400 × g. Eluate was transferred to a fresh 1.5 ml tube and neutralised with 3 µl 1.5 M Tris.HCl (pH 8.8) prior to SDS-PAGE. Antibodies used in this study were: FLAG – Sigma F3165, WTAP Santa Cruz sc-374280 (used for Fig. 1A), WTAP Abcam Ab195380 used for remaining Figures, KIAA1429 – Proteintech 25712-1-AP, METTL3 – Proteintech 15073-1-AP, HNRNPA1 – Millipore 04-1469, METTL14 – Sigma HPA038002, ACTIN – Sigma A5060, YTHDC1 – Proteintech 14392-1-AP, SSRP1 – Biolegend 609701, TUBULIN – Sigma T5168, ALYREF – Abcam Ab6141, NXF1 – Abcam Ab50609, DDX39A/B and THOC5 were described previously^{3 (link)}.