Protein g magnetic beads

Palmitic acid (#P5585) and fatty acid-free BSA (#A3803) were obtained from Sigma-Aldrich, St. Louis, MO. Magnetic protein G beads (#S1430S) were purchased from New England Bio Labs, Ipswich, MA. Trizol reagent and all other chemicals were obtained from Thermo Fisher Scientific, Waltham, MA.

20 μg of antibodies were digested with 120 U of IDEZ (NEB) for 1 hour at 37°C to separate the F(ab′)₂ and Fc regions. The Fc region was purified by incubating digested antibodies with magnetic protein G beads (NEB) for an additional hour at room temperature. Beads were washed with 2X with distilled water. Beads were then incubated with PNGaseF (ThermoFisher Scientific) to remove the N-linked glycan at 50°C for 1 hour. Released glycans were purified and labeled using the GlycanAssure APTS labeling kit (ThermoFisher Scientific) according to manufacturer’s instructions. Labeled glycans were analyzed on a 3500xL Genetic Analyzer (Applied Biosystems) using a POP7 polymer. Glycan peaks and relative abundance of glycan content was analyzed using the GlycanAssure Data Analysis Software v1.0 (Applied Biosystems).

K1 or BCPAP cells stably overexpressing METTL16 or with METTL16 knocked down and their corresponding control cells were crosslinked by UV (260 nm, 130 mJ/cm²) and harvested in cold PBS. RNA immunoprecipitation (RIP) was performed using a Magna RIP Kit (17–700, Millipore). Briefly, harvested cells were lysed (10% for input) and incubated with an anti-YTHDC2 antibody (1:1000; ab220160; Abcam) overnight at 4 °C. After washing, the immunoprecipitated complex was digested with proteinase K. RNA was extracted, detected via qRT‒PCR and normalized to the input. For m⁶A RNA binding experiments, total RNA from K1 cells stably overexpressing METTL16 or with METTL16 knocked down was treated with deoxyribonuclease I (Solarbio, China). The RNAs were sonicated and precipitated with Protein G Magnetic Beads (S1430S, NEB) bound to a m⁶A antibody (202,003, SYSY). After proteinase K (10 µg/mL) enzymolysis, RNAs were isolated for qRT‒PCR analysis (the input served as a control). For m⁶A sequencing, m⁶A-RNAs were acquired from the aforementioned m⁶A-RIP assay. The RNA libraries were created via quality inspection and subsequently subjected to analysis on an Illumina HiSeq instrument. The peaks were visualized with IGV software.

Cells were seeded at 500,000 in 60 mm plates the day before transfection. Transfections were performed in duplicate with 2.5 μg of each plasmid using either JetPEI or Lipo2000. 2 plates per transfection were harvested 48–72 h later in 1 ml NEB lysis buffer (150 mM NaCl, 10 mMTris.Cl pH7.4, 1 mM EDTA, 1 mM EGTA, 1% TritonX100, 0.5% NP40 supplemented with Roche Ultra protease inhibitors, PefablocSC, Phosphatase inhibitors). The samples were sonicated and then rotated in the cold room for 30 minutes, before centrifugation at 4 °C for 10 minutes at 20,000 ×  g. A concentration of 20 μg of protein was removed for input analysis. Equivalent quantities of protein were used for the immunoprecipitations. To avoid detecting antibodies during the western blot, 2 μg of antibody (anti-HA 3F10 Roche) was crosslinked to either Protein A or Protein G magnetic beads (NEB) using dimethyl pimelimidate dihydrochloride (D8388 SIGMA). After 30 min preclearing, 25 μl of crosslinked beads were added to each lysate and incubated with rotation in the cold room for a minimum time of 1 h. Beads were washed 3Xs with NEB buffer. Proteins were released from the beads using 25 μl of 0.1 M glycine pH2.5. 5 μl of 1 M Tris.Cl pH7.5 was added to neutralize the acidity. Samples were charged with Laemmli loading buffer/β−mercaptomethanol after heating at 70 °C for 10 min.