Abstract We recently developed Di rected Me thylation with Lo ng-read seq uencing (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 days for performing in situ targeted methylation, 1-5 days for library preparation depending on desired fragment length, and 1-3 days 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. Key papers Altemose, N., Maslan, A., Smith, O.K., Sundararajan, K., Brown, R.R., Mishra, R., Detweiler, A.M., Neff, N., Miga, K.H., Straight, A.F. and Streets, A., 2022. DiMeLo-seq: a long-read, single-molecule method for mapping protein–DNA interactions genome wide. Nature Methods , pp.1-13. ( https://doi.org/10.1038/s41592-022-01475-6 )

Mapping protein-DNA interactions with DiMeLo-seq