Mapping protein-DNA interactions with DiMeLo-seq

We recently developed directed methylation with long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype-specific protein-DNA interactions and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule DNA sequencing platforms such as Nanopore sequencing. Here we present a detailed protocol and practical guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed or frozen cells. The protocol requires 1-2 d for performing in situ targeted methylation, 1-5 d for library preparation depending on desired fragment length and 1-3 d for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data.