Multiplexed Chromatin Immunoprecipitation Sequencing for Quantitative Study of Histone Modifications and Chromatin Factors

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2024 Oct 3

PMID 39363107

Authors

Banushree Kumar

Carmen Navarro

Philip Yuk Kwong Yung

Jing Lyu

Angelo Salazar Mantero

Anna-Maria Katsori

Hannah Schwammle

Marcel Martin

Simon J Elsasser

Affiliations

Soon will be listed here.

Abstract

ChIP-seq is a widely used technique for studying histone post-translational modifications and DNA-binding proteins. DNA fragments associated with a specific protein or histone modification epitope are captured by using antibodies, sequenced and mapped to a reference genome. Albeit versatile and popular, performing many parallel ChIP-seq experiments to compare different conditions, replicates and epitopes is laborious, is prone to experimental variation and does not allow quantitative comparisons unless adequate spike-in chromatin is included. We present a detailed protocol for performing and analyzing a multiplexed quantitative chromatin immunoprecipitation-sequencing experiment (MINUTE-ChIP), in which multiple samples are profiled against multiple epitopes in a single workflow. Multiplexing not only dramatically increases the throughput of ChIP-seq experiments (e.g., profiling 12 samples against multiple histone modifications or DNA-binding proteins in a single experiment), but also enables accurate quantitative comparisons. The protocol consists of four parts: sample preparation (i.e., lysis, chromatin fragmentation and barcoding of native or formaldehyde-fixed material), pooling and splitting of the barcoded chromatin into parallel immunoprecipitation reactions, preparation of next-generation sequencing libraries from input and immunoprecipitated DNA and data analysis using our dedicated analysis pipeline. This pipeline autonomously generates quantitatively scaled ChIP-seq tracks for downstream analysis and visualization, alongside necessary quality control indicators. The entire workflow requires basic knowledge in molecular biology and bioinformatics and can be completed in 1 week. MINUTE-ChIP empowers biologists to perform every ChIP-seq experiment with an appropriate number of replicates and control conditions, delivering more statistically robust, exquisitely quantitative and biologically meaningful results.

References

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

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

Johnson D, Mortazavi A, Myers R, Wold B . Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007; 316(5830):1497-502. DOI: 10.1126/science.1141319. View

Mikkelsen T, Ku M, Jaffe D, Issac B, Lieberman E, Giannoukos G . Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007; 448(7153):553-60. PMC: 2921165. DOI: 10.1038/nature06008. View

Cuddapah S, Barski A, Cui K, Schones D, Wang Z, Wei G . Native chromatin preparation and Illumina/Solexa library construction. Cold Spring Harb Protoc. 2010; 2009(6):pdb.prot5237. PMC: 3541822. DOI: 10.1101/pdb.prot5237. View

ONeill L, Turner B . Immunoprecipitation of native chromatin: NChIP. Methods. 2003; 31(1):76-82. DOI: 10.1016/s1046-2023(03)00090-2. View

Kasinathan S, Orsi G, Zentner G, Ahmad K, Henikoff S . High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods. 2013; 11(2):203-9. PMC: 3929178. DOI: 10.1038/nmeth.2766. View

Jin H, Kasper L, Larson J, Wu G, Baker S, Zhang J . ChIPseqSpikeInFree: a ChIP-seq normalization approach to reveal global changes in histone modifications without spike-in. Bioinformatics. 2019; 36(4):1270-1272. PMC: 7523640. DOI: 10.1093/bioinformatics/btz720. View

Chen K, Hu Z, Xia Z, Zhao D, Li W, Tyler J . The Overlooked Fact: Fundamental Need for Spike-In Control for Virtually All Genome-Wide Analyses. Mol Cell Biol. 2015; 36(5):662-7. PMC: 4760223. DOI: 10.1128/MCB.00970-14. View

10.

Orlando D, Chen M, Brown V, Solanki S, Choi Y, Olson E . Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep. 2014; 9(3):1163-70. DOI: 10.1016/j.celrep.2014.10.018. View

11.

Blanco E, Di Croce L, Aranda S . SpikChIP: a novel computational methodology to compare multiple ChIP-seq using spike-in chromatin. NAR Genom Bioinform. 2021; 3(3):lqab064. PMC: 8315120. DOI: 10.1093/nargab/lqab064. View

12.

Grzybowski A, Chen Z, Ruthenburg A . Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Mol Cell. 2015; 58(5):886-99. PMC: 4458216. DOI: 10.1016/j.molcel.2015.04.022. View

13.

Lara-Astiaso D, Weiner A, Lorenzo-Vivas E, Zaretsky I, Jaitin D, David E . Immunogenetics. Chromatin state dynamics during blood formation. Science. 2014; 345(6199):943-9. PMC: 4412442. DOI: 10.1126/science.1256271. View

14.

van Galen P, Viny A, Ram O, Ryan R, Cotton M, Donohue L . A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes. Mol Cell. 2015; 61(1):170-80. PMC: 4707994. DOI: 10.1016/j.molcel.2015.11.003. View

15.

Chabbert C, Adjalley S, Steinmetz L, Pelechano V . Multiplexed ChIP-Seq Using Direct Nucleosome Barcoding: A Tool for High-Throughput Chromatin Analysis. Methods Mol Biol. 2017; 1689:177-194. DOI: 10.1007/978-1-4939-7380-4_16. View

16.

Arrigoni L, Al-Hasani H, Ramirez F, Panzeri I, Ryan D, Santacruz D . RELACS nuclei barcoding enables high-throughput ChIP-seq. Commun Biol. 2018; 1:214. PMC: 6281648. DOI: 10.1038/s42003-018-0219-z. View

17.

Kumar B, Elsasser S . Quantitative Multiplexed ChIP Reveals Global Alterations that Shape Promoter Bivalency in Ground State Embryonic Stem Cells. Cell Rep. 2019; 28(12):3274-3284.e5. PMC: 6859498. DOI: 10.1016/j.celrep.2019.08.046. View

18.

Kumar B, Navarro C, Winblad N, Schell J, Zhao C, Weltner J . Polycomb repressive complex 2 shields naïve human pluripotent cells from trophectoderm differentiation. Nat Cell Biol. 2022; 24(6):845-857. PMC: 9203276. DOI: 10.1038/s41556-022-00916-w. View

19.

Weigert R, Hetzel S, Bailly N, Haggerty C, Ilik I, Yung P . Dynamic antagonism between key repressive pathways maintains the placental epigenome. Nat Cell Biol. 2023; 25(4):579-591. PMC: 10104784. DOI: 10.1038/s41556-023-01114-y. View

20.

Shao R, Kumar B, Lidschreiber K, Lidschreiber M, Cramer P, Elsasser S . Distinct transcription kinetics of pluripotent cell states. Mol Syst Biol. 2022; 18(1):e10407. PMC: 8754154. DOI: 10.15252/msb.202110407. View