Rna purification column

Total RNA was extracted from cells overexpressing PRMT6 or YTHDF2. The extracted RNA was fragmented using RNA Fragmentation Reagents (Invitrogen, Cat#AM8740) to approximately 300nt in size, and fragmented RNA was recovered using an RNA purification column (Zymo Research, Cat#R1017). BSA was mixed with Protein A/G magnetic beads and rotated at 4 °C for 2 h to block the beads. 50 µg of fragmented RNA was then mixed with 5 µg of m⁶A antibody or IgG control and incubated with the blocked beads overnight at 4 °C. After collecting the beads with a magnetic stand and discarding the supernatant, the bound RNA was eluted and digested with proteinase K. The eluted RNA was extracted using an RNA purification column, reverse transcribed, and quantitatively analyzed by qPCR. Primers for MeRIP experiments are listed in Supplementary Material 8: Table S2.

For RNA preparation approximately 500 animals were grown at 20°C on empty vector or cye-1(RNAi) plates for 4 days starting from L4 stage. Worms were washed in M9 buffer to remove bacteria. RNA was extracted with TriReagent (Sigma), treated with DNAse (Quiagen), and purified using a RNA purification column (ZYMO Research). RNA concentration and quality was assessed using a NanoPhotometer P-Class. cDNA was synthesized with reverse transcriptase (SuperScript III First-Strand Synthesis System, Invitrogen) and oligo-dT primer. qPCR runs were per-formed in technical triplicates on a Roche Light Cycler 480 using the Takyon No Rox SYBR MasterMix blue dTTP (Eurogentec).
Samples were analyzed by the 2^−ΔΔCt method with normalization to the geometric mean of the reference genes cdc-42 and Y45F10D.4. At least three biological replicates were examined for each sample. Primer sequences are listed in Supplementary Table S7.