M6a methylation antibody

Total RNA was extracted, and 100 ng of RNA was spotted onto a nylon membrane (Sigma-Aldrich, GERPN1210B). Then, the membranes were UV-crosslinked and blocked with PBST (PBS with 0.1% Tween 20) containing 5% nonfat milk for 1 h. After that, the membranes were incubated with m6A methylation antibody (Synaptic Systems, 202003, 1:1000) at 4 °C overnight. Then, the membranes were washed with PBST and incubated with HRP-conjugated secondary antibodies (1:3,000, Boster, BA1054) for 1 h at room temperature. The membranes were washed with PBST and detected by using Immobilon Western Chemiluminescent HRP Substrate (Millipore WBKLS0500).
The same amount of RNA was spotted on another nylon membrane, and UV crosslinking was performed. Then, the membranes were stained with 0.02% methylene blue solution (pH 5.2) for 1 h. The membranes were washed with ribonuclease-free water for 2 h, and the results were captured by a camera.

Total RNA was extracted using TRIzol reagent following the manufacturer’s procedure. The total RNA quality and quantity were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, 5067–1511) with RIN > 7.0. Approximately more than 200 µg of total RNA was subjected to isolation of poly(A) mRNA with poly-T oligo-attached magnetic beads. Following purification, the poly(A) mRNA fractions were fragmented into ~100-nt oligonucleotides using divalent cations under an elevated temperature. Then, the cleaved RNA fragments were incubated for 2 h at 4 °C with m6A methylation antibody (Synaptic Systems, 202003) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) with BSA (0.5 μg/μl). The mixture was then incubated with Protein A beads and eluted with elution buffer (1× IP buffer and 6.7 mM m6A). Eluted RNA was precipitated with 75% ethanol. Eluted m6A-containing fragments (IP) and untreated input control fragments were converted to a final cDNA library in accordance with strand-specific library preparation by the dUTP method. The average insert size for the paired-end libraries was ~100 ± 50 bp. Then, we performed paired-end 2 × 150 bp sequencing on an Illumina Novaseq™ 6000 platform at LC-BIO Biotech, Ltd. (Hangzhou, China), following the vendor’s recommended protocol.

RIP was performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, 17–701) following the manufacturer's instructions. MSCs were lysed with RIPA buffer and incubated with magnetic beads conjugated with specific antibodies, including m6A methylation antibody (Synaptic Systems, 202003, 1:100), anti-IGF2BP3 (Abcam, ab177477, 1:100), anti-METTL3 (Abcam, ab195352, 1:50), anti-FTO (Abcam, ab177477, 1:100), or negative control IgG (Santa Cruz, sc-3877, 1:100). The immunoprecipitated RNA was then extracted and reverse-transcribed. Finally, the abundance of MYLK mRNA was measured by RT-qPCR.