Tissue-specific DamID protocol using nanopore sequencing


  • Georgina Gómez-Saldivar Department of Biology, University of Fribourg, Switzerland
  • Dominique A. Glauser Department of Biology, University of Fribourg, Switzerland
  • Peter Meister Cell Fate and Nuclear Organization, Institute of Cell Biology, University of Bern, Switzerland


CRE recombinase-based tissue-specific expression, DNA adenine methylation identification, DNA-protein interaction, long-read sequencing, third generation sequencing


DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of E. coli, which methylates proximal DNA in vivo. Here, we present an optimized procedure, which was developed for tissue-specific analyses in Caenorhabditis elegans and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details C. elegans-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.


Kaletsky R, Yao V, Williams A, Runnels AM, Tadych A, Zhou S, et al. Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression. PLoS Genet. 2018 Aug;14(8):e1007559. https://doi.org/10.1371/journal.pgen.1007559 PMID:30096138

Haenni S, Ji Z, Hoque M, Rust N, Sharpe H, Eberhard R, et al. Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3′-end-seq. Nucleic Acids Res. 2012 Jul;40(13):6304–18. https://doi.org/10.1093/nar/gks282 PMID:22467213

Ma X, Zhan G, Sleumer MC, Chen S, Liu W, Zhang MQ, et al. Analysis of C. elegans muscle transcriptome using trans-splicing-based RNA tagging (SRT). Nucleic Acids Res. 2016 Dec;44(21):e156. PMID:27557708

Blazie SM, Babb C, Wilky H, Rawls A, Park JG, Mangone M. Comparative RNA-Seq analysis reveals pervasive tissue-specific alternative polyadenylation in Caenorhabditis elegans intestine and muscles. BMC Biol. 2015 Jan;13(1):4. https://doi.org/10.1186/s12915-015-0116-6 PMID:25601023

van Steensel B, Henikoff S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol. 2000 Apr;18(4):424–8. https://doi.org/10.1038/74487 PMID:10748524

Woolcock KJ, Gaidatzis D, Punga T, Bühler M. Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe. Nat Struct Mol Biol. 2011 Jan;18(1):94–9. https://doi.org/10.1038/nsmb.1935 PMID:21151114

Steglich B, Filion GJ, van Steensel B, Ekwall K. The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe. Nucleus. 2012 Jan-Feb;3(1):77–87. https://doi.org/10.4161/nucl.18825 PMID:22156748

McCann TS, Guo Y, McDonald WH, Tansey WP. Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing. Proc Natl Acad Sci USA. 2016 Feb;113(5):1309–14. https://doi.org/10.1073/pnas.1518375113 PMID:26787877

Flury V, Georgescu PR, Iesmantavicius V, Shimada Y, Kuzdere T, Braun S, et al. The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1. Mol Cell. 2017 Jul;67(2):294–307.e9. https://doi.org/10.1016/j.molcel.2017.05.026 PMID:28648780

Spasskaya DS, Nadolinskaia NI, Tutyaeva VV, Lysov YP, Karpov VL, Karpov DS. Yeast Rpn4 Links the Proteasome and DNA Repair via RAD52 Regulation. Int J Mol Sci. 2020 Oct;21(21):E8097. https://doi.org/10.3390/ijms21218097 PMID:33143019

Towbin BD, González-Aguilera C, Sack R, Gaidatzis D, Kalck V, Meister P, et al. Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery. Cell. 2012 Aug;150(5):934–47. https://doi.org/10.1016/j.cell.2012.06.051 PMID:22939621

Sharma R, Jost D, Kind J, Gómez-Saldivar G, van Steensel B, Askjaer P, et al. Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans. Genes Dev. 2014 Dec;28(23):2591–6. https://doi.org/10.1101/gad.248864.114 PMID:25452271

Schuster E, McElwee JJ, Tullet JM, Doonan R, Matthijssens F, Reece-Hoyes JS, et al. DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO. Mol Syst Biol. 2010 Aug;6(1):399. https://doi.org/10.1038/msb.2010.54 PMID:20706209

Van de Walle P, Muñoz-Jiménez C, Askjaer P, Schoofs L, Temmerman L. DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans. PLoS One. 2020 Dec;15(12):e0242939. https://doi.org/10.1371/journal.pone.0242939 PMID:33306687

Muñoz-Jiménez C, Ayuso C, Dobrzynska A, Torres-Mendéz A, Ruiz PC, Askjaer P. An Efficient FLP-Based Toolkit for Spatiotemporal Control of Gene Expression in Caenorhabditis elegans. Genetics. 2017 Aug;206(4):1763–78. https://doi.org/10.1534/genetics.117.201012 PMID:28646043

Cabianca DS, Muñoz-Jiménez C, Kalck V, Gaidatzis D, Padeken J, Seeber A, et al. Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei. Nature. 2019 May;569(7758):734–9. https://doi.org/10.1038/s41586-019-1243-y PMID:31118512

Harr JC, Schmid CD, Muñoz-Jiménez C, Romero-Bueno R, Kalck V, Gonzalez-Sandoval A, et al. Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model. Genes Dev. 2020 Apr;34(7-8):560–79. https://doi.org/10.1101/gad.332213.119 PMID:32139421

Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van Steensel B. Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet. 2006 Sep;38(9):1005–14. https://doi.org/10.1038/ng1852 PMID:16878134

Orian A, van Steensel B, Delrow J, Bussemaker HJ, Li L, Sawado T, et al. Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev. 2003 May;17(9):1101–14. https://doi.org/10.1101/gad.1066903 PMID:12695332

Ilyin AA, Ryazansky SS, Doronin SA, Olenkina OM, Mikhaleva EA, Yakushev EY, et al. Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells. Nucleic Acids Res. 2017 Jul;45(13):7666–80. https://doi.org/10.1093/nar/gkx355 PMID:28472469

Flint Brodsly N, Bitman-Lotan E, Boico O, Shafat A, Monastirioti M, Gessler M, et al. The transcription factor Hey and nuclear lamins specify and maintain cell identity. eLife. 2019 Jul;8:e44745. https://doi.org/10.7554/eLife.44745 PMID:31310235

Estacio-Gómez A, Hassan A, Walmsley E, Le LW, Southall TD. Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development. Biol Open. 2020 Jun;9(5):bio052928. https://doi.org/10.1242/bio.052928 PMID:32493733

Hassan A, Araguas Rodriguez P, Heidmann SK, Walmsley EL, Aughey GN, Southall TD. Condensin I subunit Cap-G is essential for proper gene expression during the maturation of post-mitotic neurons. eLife. 2020 Apr;9:e55159. https://doi.org/10.7554/eLife.55159 PMID:32255428

Gutierrez-Triana JA, Mateo JL, Ibberson D, Ryu S, Wittbrodt J. iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins. Development. 2016 Nov;143(22):4272–8. https://doi.org/10.1242/dev.139261 PMID:27707796

Germann S, Juul-Jensen T, Letarnec B, Gaudin V. DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci. Plant J. 2006 Oct;48(1):153–63. https://doi.org/10.1111/j.1365-313X.2006.02859.x PMID:16972870

Bollier N, Sicard A, Leblond J, Latrasse D, Gonzalez N, Gévaudant F, et al. At-MINI ZINC FINGER2 and Sl-INHIBITOR OF MERISTEM ACTIVITY, a Conserved Missing Link in the Regulation of Floral Meristem Termination in Arabidopsis and Tomato. Plant Cell. 2018 Jan;30(1):83–100. https://doi.org/10.1105/tpc.17.00653 PMID:29298836

Alvarez JM, Schinke AL, Brooks MD, Pasquino A, Leonelli L, Varala K, et al. Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade. Nat Commun. 2020 Mar;11(1):1157. https://doi.org/10.1038/s41467-020-14979-6 PMID:32123177

Tosti L, Ashmore J, Tan BS, Carbone B, Mistri TK, Wilson V, et al. Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo. Genome Res. 2018 Apr;28(4):592–605. https://doi.org/10.1101/gr.227124.117 PMID:29572359

Szczesnik T, Ho JW, Sherwood R. Dam mutants provide improved sensitivity and spatial resolution for profiling transcription factor binding. Epigenetics Chromatin. 2019 Jun;12(1):36. https://doi.org/10.1186/s13072-019-0273-x PMID:31196130

Gatticchi L, de Las Heras JI, Roberti R, Schirmer EC. Optimization of DamID for use in primary cultures of mouse hepatocytes. Methods. 2019 Mar;157:88–99. https://doi.org/10.1016/j.ymeth.2018.11.005 PMID:30445179

Yattah C, Hernandez M, Huang D, Park H, Liao W, Casaccia P. Dynamic Lamin B1-Gene Association During Oligodendrocyte Progenitor Differentiation. Neurochem Res. 2020 Mar;45(3):606–19. https://doi.org/10.1007/s11064-019-02941-y PMID:32020491

Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, et al. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature. 2008 Jun;453(7197):948–51. https://doi.org/10.1038/nature06947 PMID:18463634

Vogel MJ, Guelen L, de Wit E, Peric-Hupkes D, Lodén M, Talhout W, et al. Human heterochromatin proteins form large domains containing KRAB-ZNF genes. Genome Res. 2006 Dec;16(12):1493–504. https://doi.org/10.1101/gr.5391806 PMID:17038565

Robson MI, de Las Heras JI, Czapiewski R, Sivakumar A, Kerr AR, Schirmer EC. Constrained release of lamina-associated enhancers and genes from the nuclear envelope during T-cell activation facilitates their association in chromosome compartments. Genome Res. 2017 Jul;27(7):1126–38. https://doi.org/10.1101/gr.212308.116 PMID:28424353

de Las Heras JI, Zuleger N, Batrakou DG, Czapiewski R, Kerr AR, Schirmer EC. Tissue-specific NETs alter genome organization and regulation even in a heterologous system. Nucleus. 2017 Jan;8(1):81–97. https://doi.org/10.1080/19491034.2016.1261230 PMID:28045568

Aughey GN, Estacio Gomez A, Thomson J, Yin H, Southall TD. CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo. eLife. 2018 Feb;7:e32341. https://doi.org/10.7554/eLife.32341 PMID:29481322

Kind J, Pagie L, Ortabozkoyun H, Boyle S, de Vries SS, Janssen H, et al. Single-cell dynamics of genome-nuclear lamina interactions. Cell. 2013 Mar;153(1):178–92. https://doi.org/10.1016/j.cell.2013.02.028 PMID:23523135

Cheetham SW, Gruhn WH, van den Ameele J, Krautz R, Southall TD, Kobayashi T, et al. Targeted DamID reveals differential binding of mammalian pluripotency factors. Development. 2018 Oct;145(20):dev170209. https://doi.org/10.1242/dev.170209 PMID:30185410

Hass MR, Liow HH, Chen X, Sharma A, Inoue YU, Inoue T, et al. SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers. Mol Cell. 2016 Oct;64(1):213. https://doi.org/10.1016/j.molcel.2016.09.035 PMID:27716485

Cheetham SW, Brand AH. RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites. Nat Struct Mol Biol. 2018 Jan;25(1):109–14. https://doi.org/10.1038/s41594-017-0006-4 PMID:29323275

Redolfi J, Zhan Y, Valdes-Quezada C, Kryzhanovska M, Guerreiro I, Iesmantavicius V, et al. DamC reveals principles of chromatin folding in vivo without crosslinking and ligation. Nat Struct Mol Biol. 2019 Jun;26(6):471–80. https://doi.org/10.1038/s41594-019-0231-0 PMID:31133702

Southall TD, Gold KS, Egger B, Davidson CM, Caygill EE, Marshall OJ, et al. Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells. Dev Cell. 2013 Jul;26(1):101–12. https://doi.org/10.1016/j.devcel.2013.05.020 PMID:23792147

Gómez-Saldivar G, Osuna-Luque J, Semple JI, Glauser DA, Jarriault S, Meister P. Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans. Genetics. 2020 Dec;216(4):931–45. https://doi.org/10.1534/genetics.120.303774 PMID:33037050

Rooijers K, Markodimitraki CM, Rang FJ, de Vries SS, Chialastri A, de Luca KL, et al. Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells. Nat Biotechnol. 2019 Jul;37(7):766–72. https://doi.org/10.1038/s41587-019-0150-y PMID:31209373

Markodimitraki CM, Rang FJ, Rooijers K, de Vries SS, Chialastri A, de Luca KL, et al. Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq. Nat Protoc. 2020 Jun;15(6):1922–53. https://doi.org/10.1038/s41596-020-0314-8 PMID:32350457

van Dijk EL, Jaszczyszyn Y, Naquin D, Thermes C. The Third Revolution in Sequencing Technology. Trends Genet. 2018 Sep;34(9):666–81. https://doi.org/10.1016/j.tig.2018.05.008 PMID:29941292

Ramialison M, Waardenberg AJ, Schonrock N, Doan T, de Jong D, Bouveret R, et al. Analysis of steric effects in DamID profiling of transcription factor target genes. Genomics. 2017 Mar;109(2):75–82. https://doi.org/10.1016/j.ygeno.2017.01.006 PMID:28189763

Sha K, Gu SG, Pantalena-Filho LC, Goh A, Fleenor J, Blanchard D, et al. Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans. BMC Genomics. 2010 Aug;11(1):465. https://doi.org/10.1186/1471-2164-11-465 PMID:20691096

Gómez-Saldivar G, Meister P, Askjaer P, Dam ID. DamID Analysis of Nuclear Organization in Caenorhabditis elegans. Methods Mol Biol. 2016;1411:341–58. https://doi.org/10.1007/978-1-4939-3530-7_22 PMID:27147053

Frøkjær-Jensen C, Davis MW, Sarov M, Taylor J, Flibotte S, LaBella M, et al. Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat Methods. 2014 May;11(5):529–34. https://doi.org/10.1038/nmeth.2889 PMID:24820376

Ruijtenberg S, van den Heuvel S. G1/S Inhibitors and the SWI/SNF Complex Control Cell-Cycle Exit during Muscle Differentiation. Cell. 2015 Jul;162(2):300–13. https://doi.org/10.1016/j.cell.2015.06.013 PMID:26144318

Kage-Nakadai E, Imae R, Suehiro Y, Yoshina S, Hori S, Mitani S. A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination. PLoS One. 2014 Dec;9(12):e114680. https://doi.org/10.1371/journal.pone.0114680 PMID:25474529

Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018 Sep;34(18):3094–100. https://doi.org/10.1093/bioinformatics/bty191 PMID:29750242

Marshall OJ, Brand AH. damidseq_pipeline: an automated pipeline for processing DamID sequencing datasets. Bioinformatics. 2015 Oct;31(20):3371–3. https://doi.org/10.1093/bioinformatics/btv386 PMID:26112292



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Gómez-Saldivar G, Glauser DA, Meister P. Tissue-specific DamID protocol using nanopore sequencing. J Biol Methods [Internet]. 2021Aug.27 [cited 2021Dec.4];8(3):e152. Available from: https://jbmethods.org/jbm/article/view/362