Sequencing DNA methylation and hydroxymethylation at co-occurring chromatin features

Epigenetic modifications govern chromatin dynamics and cell state. However, current methods cannot simultaneously resolve the presence of multiple DNA modifications at co-occurring chromatin-associated features. It is thus not clear how these features are physically coupled and how their combinations regulate genome function. To address this key question, we report 6-base-CUT&Tag, a method for simultaneous 6-base sequencing at target chromatin features. Using 6-base-CUT&Tag to profile 5mC and 5hmC at co-occurring histone modifications in mouse embryonic stem cells, we identify histone mark-specific signatures of methylation and hydroxymethylation to improve discrimination of different functional enhancer states. Overall design: NGS sequencing DNA libraries prepared from genomic DNA from mouse ESC cells. Cells were probed using the CUT&Tag methodology using different primary antibodies against a panel of histone marks and 6-base-seq DNA libraries were prepared from the extracted gDNA (6-base-CUT&Tag). 2-3 biological replicates are included per experiment, originated from independent E14Tg2a mESC cultures. Libraries prepared from total tagmented genomic DNA from E14 cells are also included as whole-genome (WG) controls.