Abstract
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.
Key points
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DiMeLo-seq uses long-read, single-molecule sequencing to map protein–DNA interactions genome wide, in nuclei from fresh, fixed or frozen cells, and from primary tissues or intact organisms.
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Compared with short-read methods, this enables mapping of 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.
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Data availability
Data generated for this protocol: raw sequencing data are available in the Sequence Read Archive (SRA) under BioProject accession PRJNA855257 and processed data are available on Gene Expression Omnibus (GEO) under accession GSE208125. All raw fast5 sequencing data from the accompanying Altemose et al. manuscript are available in the SRA under BioProject accession PRJNA752170. External data sources used in this protocol: H3K27ac ChIP-seq data in GM12878 available from ENCODE Project Consortium under accession ENCFF218QBO (https://www.encodeproject.org/files/ENCFF218QBO/). H3K27me3 ChIP-seq data in GM12878 available from ENCODE Project Consortium under accession ENCFF119CAV (https://www.encodeproject.org/files/ENCFF119CAV/). H3K4me3 ChIP-seq data in GM12878 available from ENCODE Project Consortium under accession ENCFF228TWF (https://www.encodeproject.org/files/ENCFF228TWF/). H3K27ac CUT&Tag data in GM12878 available on Gene Expression Omnibus (GEO) under accession GSM5530639. H3K27me3 CUT&Tag data in GM12878 available on GEO under accession GSM5530673. ATAC-seq data in GM12878 available from ENCODE Project Consortium under accession ENCFF603BJO (https://www.encodeproject.org/files/ENCFF603BJO/). TSS and gene annotations from NCBI RefSeq downloaded from UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgTrackUi?g=refSeqComposite&db=hg38). RNA-seq data in GM12878 available from ENCODE Project Consortium under accession ENCFF978HIY (https://www.encodeproject.org/files/ENCFF978HIY/). D. melanogaster H3K9me3 ChIP-seq data available on GEO under accession GSE140539. File GSE140539_H3K9me3_sorted_deepnorm_log2_smooth.bw was used.
Code availability
The dimelo Python package for analysis of DiMeLo-seq data is available on Github: https://github.com/streetslab/dimelo.
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Acknowledgements
Research reported in this publication was supported by the Chan Zuckerberg Biohub, San Francisco, and by the National Human Genome Research Institute and the National Institute of General Medical Sciences of the National Institutes of Health under award number R01HG012383 to A.S., R01GM074728 to A.F.S. and R35GM139653 to G.K. A.M. is supported by a NSF GRFP award. L.D.B. is supported by Volkswagen Stiftung (98196). N.A. is an HHMI Hanna H. Gray Fellow. A.S. is a Chan Zuckerberg Biohub Investigator and a Pew Scholar in the Biomedical Sciences. The sequencing was carried out by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01. This project has been made possible in part by grant number 2022-253563 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation.
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A.M., N.A. and A.S. designed the study. A.M., N.A. and L.D.B. performed the experiments. A.M., R.M. and J.M. developed dimelo software package. A.M. and N.A. analyzed and interpreted the data. A.M., N.A. and R.M. made the figures. A.M. and J.M. wrote the manuscript, with input from N.A., R.M., L.D.B., K.S., G.K., A.F.S. and A.S. A.S. and N.A. supervised the study.
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N.A., A.M., K.S., A.F.S. and A.S. are co-inventors on a patent application related to this work. The remaining authors declare no competing interests.
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Altemose, N. et al. Nat. Methods 19, 711–723 (2022): https://doi.org/10.1038/s41592-022-01475-6
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Maslan, A., Altemose, N., Marcus, J. et al. Mapping protein–DNA interactions with DiMeLo-seq. Nat Protoc 19, 3697–3720 (2024). https://doi.org/10.1038/s41596-024-01032-9
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DOI: https://doi.org/10.1038/s41596-024-01032-9
