A bisulfite-free approach for base-resolution analysis of genomic 5-carboxylcytosine

Due to an extreme rarity of 5-carboxylcytosine (5caC) in mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental/computational resources increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high resolution mapping of 5caCGs. The method employs an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC generating unmodified CGs which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supported the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflected their distinct demethylation dynamics and demonstrated an association of 5caC with gene expression. Generally, we demonstrated that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC. Overall design: 5-carboxylcytosine analysis in mouse embryonic stem cells. DNA modifications were analysed in the naive and primed pluripotency states - two techinal replicates for each input. Serum triple Tet knockout DNA was used as a negative control and prepared identically with other samples.