MeRIP seq of AHCY Vector and FTO SH in HCT116
Protein expression plasmids were generated by PCR amplification of the respective cDNA open reading frames. shRNAs targeting were designed using the website https://portals.broadinstitute.org/gpp/public/gene/search, cloned into the pLKO.1 plasmid (Addgene #10878). Lentiviral particles were produced in HEK293T cells using a third-generation packaging system. This system involved the utilization of PLKO.1 or PCDH plasmids, as well as psPAX2 (Addgene, 12260) and pCMV-VSV-G (Addgene, 8454) plasmids. The supernatant of transfected HEK293T cells was harvested approximately 48-72 hours later and was filtered through a 0.45 um pore size to obtain the viral stock. This viral stock couldbe stored at 4 for short-term (1-5 days) utilization or -80 for long-term storage.Total RNA was extracted according to the operation instructions (AXYGEN, AP-MN-MS-RNA-250), and the pass criteria of total RNA were as follows: concentration larger than 50ng/uL, RIN value larger than 7.0, and OD260/280 larger than 1.8. After meeting these standards, downstream experiments can be performed. mRNA was then specifically captured and purified twice through oligo(dT) magnetic beads, and was fragmented using a magnesium ion fragmentation reagent kit (NEB, E6150S). Fragmented mRNA was pre-mixed with m6A-immunomagnetic beads, and the IP product was reversely transcribed into stable cDNA. The fragment was screened and purified through magnetic beads, and a sequencing library was constructed by PCR and sequenced on a machine (LC-Bio).