m6Am-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 m6Am-exo-qPCR, the same procedure as for m6Am-exo-seq was used through the anti-m6Am 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 inSI Appendix, Table S4 .
For m6Am-exo-qPCR, the same procedure as for m6Am-exo-seq was used through the anti-m6Am 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
