TET2-mediated mRNA in tumor immunity [m5C]. TET2-mediated mRNA in tumor immunity [m5C]

To investigate the function of TET2 as an mRNA m5C demethylase in the development of acute myeloid leukemia (AML), we conducted RNA bisulfite sequencing of mRNA samples enriched from AML cells carrying Tet2+/+ versus Tet2-/- with actinomycin D treatment at different time point. Overall design: Total RNA extraction from WT and Tet2-null NRAS/AE9a cells was performed using TRIzol reagent and then was enriched for poly(A) + RNA using polyATract mRNA isolation system IV kit (Z5310, Promega) following manufacturer’s instructions. The purified RNA was then subjected to bisulfite treatment using EZ RNA Methylation Kit (R5002, Zymo Research). The converted RNA was then subjected to column-based purification and next-generation sequencing using Illumina HiSeq 2500 platform with 70 million single end reads per sample.