Fragmentation reagents kit

m⁶A_m-exo-seq was performed according to protocols developed by the Shi group (24 (link)). Briefly, mRNA was extracted from PCIF1-KO and control Calu-3 cells using a Magnetic mRNA Isolation Kit. Aliquots (100 μg) of mRNA were fragmented using a Fragmentation Reagents Kit (Invitrogen, AM8740) according to the manufacturer’s protocol. Fragmented mRNAs were phosphorylated with T4 PNK (NEB, M0201S) and then dephosphorylated with Terminator 5′-Phosphate-Dependent Exonuclease (Lucigen). Finally, Cap-Clip (CellScript) was added to remove capped transcripts. Of the final uncapped fragmented mRNA preparation, 10% was reserved as input material, and the remaining 90% was subjected to immunoprecipitation with anti-m⁶A antibody (Abcam, ab151230). Immunoprecipitated RNA was eluted with RLT buffer (QIAGEN, 160051456), purified by ethanol precipitation, and prepared for library generation using a TruSeq mRNA library preparation kit (Illumina). Sequencing was performed at IGM Genomics Center, UCSD, using an Illumina NovaSeq 6000.
For m⁶A_m-exo-qPCR, the same procedure as for m⁶A_m-exo-seq was used through the anti-m⁶A_m immunoprecipitation and RNA elution step. The eluted RNA and input samples were reverse transcribed and subjected to qPCR on a LightCycler 480 (Roche Diagnostics) using the primers listed in SI Appendix, Table S4.

m⁶Am‐exo‐Seq was performed according to previously described protocols (Sendinc et al, 2019 (link)). Briefly, mRNA was purified from control and PCIF1‐depeleted HCT116 cells using a Magnetic mRNA Isolation Kit (New England Biolabs, S1550S). mRNA was then fragmented with a Fragmentation Reagents Kit (Invitrogen, AM8740), phosphorylated with T4 PNK (NEB, M0201S), and treated with Terminator 5′‐Phosphate‐Dependent Exonuclease (Lucigen) to remove phosphorylated transcripts. Lastly, Cap‐Clip (CellScript) was used to remove capped transcripts. A sample equivalent to 10% of the 5′‐uncapped mRNA fragments was reserved as input, and the remainder was immunoprecipitated with anti‐m⁶A antibody (Abcam, ab151230). Library generation and sequencing were performed at IGM Genomics core, UCSD on an Illumina NovaSeq 6000.
For m⁶Am‐exo‐qPCR, mRNA was extracted from freshly excised control and Pcif1‐depeleted tumors and processed using the procedures described above. Reserved input and anti‐m⁶Am immunoprecipitated samples were reverse‐transcribed and analyzed by qPCR as described above.