Absolute transcription-start nucleotide m6Am stoichiometry quantification by CROWN-Seq

The 5' end RNA molecules transcribed by RNA polymerase II are known to be modified in human cells. Specifically, A-initiated transcripts can be methylated at both the 2'-O position in the ribose (2'-O-methylation, or cap-1) and the N6-position in the base (N6-methylation). Almost all A-initiated transcripts have the cap-1 modification, however, the N6-methylation is optional. As a result, A-initiated transcripts in the cell can be Am (cap-1 only) or m6Am (cap-1 and N6-methylation) modified. To study m6Am, we developed CROWN-seq, which simultaneously obtains the transcription-start information and the stoichiometry of N6-methyl. With CROWN-seq, we found that the previous m6Am mapping studies highly underestimated the diversity of the transcription-start site (nucleotide) of the transcriptome. In this study, we used CROWN-seq to reveal m6Am sites and stoichiometry among different human cell lines. We demonstrated that m6Am stoichiometry in mRNA is in general high, however, m6Am stoichiometry in snRNA, snoRNA, and their pseudogenes is more variable. Overall design: We performed both technical replicates and biological replicates of CROWN-seq in different cell lines. Technical replicates were performed by using the same purified mRNA as input.