ChIP-seq Guidelines and Practices of the ENCODE and ModENCODE Consortia

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

Citing Articles

Enhancer transcription profiling reveals an enhancer RNA-driven ferroptosis and new therapeutic opportunities in prostate cancer.

Ma S, Wang Z, Xiong Z, Ge Y, Xu M, Zhang J Signal Transduct Target Ther. 2025; 10(1):87.

PMID: 40082405 PMC: 11906896. DOI: 10.1038/s41392-025-02170-6.

A WYL domain transcription factor regulates Lactiplantibacillus plantarum intestinal colonization via perceiving c-di-GMP.

Guo Q, Wang G, Zheng L, Xue H, Wang R, Fang Y Nat Commun. 2025; 16(1):2193.

PMID: 40038299 PMC: 11880434. DOI: 10.1038/s41467-025-57581-4.

CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis.

Yang X, Cheng L, Xin Y, Zhang J, Chen X, Xu J Genome Biol. 2025; 26(1):44.

PMID: 40022213 PMC: 11869676. DOI: 10.1186/s13059-025-03510-z.

Histone lactylation-driven B7-H3 expression promotes tumor immune evasion.

Ma Z, Yang J, Jia W, Li L, Li Y, Hu J Theranostics. 2025; 15(6):2338-2359.

PMID: 39990209 PMC: 11840737. DOI: 10.7150/thno.105947.

Staphylococcus aureus induces mitophagy via the HDAC11/IL10 pathway to sustain intracellular survival.

Yang Y, Zhou H, Li F, Zhang Y, Yang J, Shen Y J Transl Med. 2025; 23(1):156.

PMID: 39905391 PMC: 11796158. DOI: 10.1186/s12967-025-06161-7.

References

Zhong M, Niu W, Lu Z, Sarov M, Murray J, Janette J . Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response. PLoS Genet. 2010; 6(2):e1000848. PMC: 2824807. DOI: 10.1371/journal.pgen.1000848. View

Lin Y, Jhunjhunwala S, Benner C, Heinz S, Welinder E, Mansson R . A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate. Nat Immunol. 2010; 11(7):635-43. PMC: 2896911. DOI: 10.1038/ni.1891. View

Dohm J, Lottaz C, Borodina T, Himmelbauer H . Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 2008; 36(16):e105. PMC: 2532726. DOI: 10.1093/nar/gkn425. View

Iyer V, Horak C, Scafe C, Botstein D, Snyder M, Brown P . Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature. 2001; 409(6819):533-8. DOI: 10.1038/35054095. View

Park P . ChIP-seq: advantages and challenges of a maturing technology. Nat Rev Genet. 2009; 10(10):669-80. PMC: 3191340. DOI: 10.1038/nrg2641. View

Ren B, Robert F, Wyrick J, Aparicio O, Jennings E, Simon I . Genome-wide location and function of DNA binding proteins. Science. 2000; 290(5500):2306-9. DOI: 10.1126/science.290.5500.2306. View

Ernst J, Kellis M . Discovery and characterization of chromatin states for systematic annotation of the human genome. Nat Biotechnol. 2010; 28(8):817-25. PMC: 2919626. DOI: 10.1038/nbt.1662. View

Celniker S, Dillon L, Gerstein M, Gunsalus K, Henikoff S, Karpen G . Unlocking the secrets of the genome. Nature. 2009; 459(7249):927-30. PMC: 2843545. DOI: 10.1038/459927a. View

Barski A, Cuddapah S, Cui K, Roh T, Schones D, Wang Z . High-resolution profiling of histone methylations in the human genome. Cell. 2007; 129(4):823-37. DOI: 10.1016/j.cell.2007.05.009. View

10.

Ernst J, Kheradpour P, Mikkelsen T, Shoresh N, Ward L, Epstein C . Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011; 473(7345):43-9. PMC: 3088773. DOI: 10.1038/nature09906. View

11.

Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T . Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007; 4(8):651-7. DOI: 10.1038/nmeth1068. View

12.

. The ENCODE (ENCyclopedia Of DNA Elements) Project. Science. 2004; 306(5696):636-40. DOI: 10.1126/science.1105136. View

13.

Valouev A, Johnson D, Sundquist A, Medina C, Anton E, Batzoglou S . Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat Methods. 2009; 5(9):829-34. PMC: 2917543. DOI: 10.1038/nmeth.1246. View

14.

Fernandez P, Frank S, Wang L, Schroeder M, Liu S, Greene J . Genomic targets of the human c-Myc protein. Genes Dev. 2003; 17(9):1115-29. PMC: 196049. DOI: 10.1101/gad.1067003. View

15.

Hua S, Kittler R, White K . Genomic antagonism between retinoic acid and estrogen signaling in breast cancer. Cell. 2009; 137(7):1259-71. PMC: 3374131. DOI: 10.1016/j.cell.2009.04.043. View

16.

Wang Z, Zang C, Rosenfeld J, Schones D, Barski A, Cuddapah S . Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet. 2008; 40(7):897-903. PMC: 2769248. DOI: 10.1038/ng.154. View

17.

DeKoter R, Singh H . Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science. 2000; 288(5470):1439-41. DOI: 10.1126/science.288.5470.1439. View

18.

Ji H, Jiang H, Ma W, Johnson D, Myers R, Wong W . An integrated software system for analyzing ChIP-chip and ChIP-seq data. Nat Biotechnol. 2008; 26(11):1293-300. PMC: 2596672. DOI: 10.1038/nbt.1505. View

19.

Lieb J, Liu X, Botstein D, Brown P . Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat Genet. 2001; 28(4):327-34. DOI: 10.1038/ng569. View

20.

Wilson N, Foster S, Wang X, Knezevic K, Schutte J, Kaimakis P . Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. Cell Stem Cell. 2010; 7(4):532-44. DOI: 10.1016/j.stem.2010.07.016. View