Nanobody-tethered transposition for dual profiling of chromatin accessibility and DNA methylation

DNA methylation and histone modifications are critical epigenetic regulators that orchestrate gene expression and modulate various physiological and pathological processes. However, existing methodologies for simultaneous profiling of these epigenetic marks often require high cell input and suffer from data loss due to bisulfite conversion. In this study, we present advanced multimodal chromatin profiling methods, MethylTag and Multi-MethylTag, that address these challenges by integrating Tn5 transposase with methylated adaptors and optimizing post-bisulfite library preparation. These methods enable high-resolution, multi-dimensional chromatin profiling with reduced cell input and improved data integrity. We validated these techniques in various human and mouse cell lines, revealing complex interactions between DNA methylation and histone modifications. Our findings highlight the utility of these approaches in enhancing epigenetic research and deepening our understanding of the regulatory mechanisms underlying gene expression. Overall design: In the 5mC-CUT&Tag method, Tn5 transposase is coupled with protein A/G (pA/G-Tn5) and preloaded with methylated P5 adaptors30. This pA/G-Tn5 complex is directed to specific histone modifications via primary and secondary antibodies, facilitating the incorporation of methylated P5 adaptors at the 5' ends of nearby DNA fragments. During the subsequent bisulfite treatment, unmethylated cytosines (C) are converted to uracils (read as thymine (T) during sequencing), while 5-methylcytosines (5mC) and the methylated adaptors remain unchanged. Post-bisulfite treatment, the ligation of the 3' adaptor ensures that the library can be efficiently amplified by PCR, which reduces the high duplication rates and information loss often caused by DNA template breakage during bisulfite conversion. Additionally, fragmented ?DNA with methylated adaptors is included as a spike-in control before bisulfite treatment to monitor the C-to-T conversion rate without affecting the amplification and sequencing.