TABLE 2.

Methods to study histone marks.

Approach	Method(s)	Description	Amount of material	Bulk or single-cell	Global pattern?	References
PCR	ChIP-qPCR	Chromatin is cross-linked, fragmented, and immunoprecipitated (ChIP). DNA is isolated and purified and undergoes PCR	5 × 105 – 5 × 106 cells	Bulk	No	Milne et al., 2009
High-throughput sequencing	ChIP-seq	Following ChIP, Next-generation sequencing (NGS) is used to identify DNA fragments and map them against entire genome	105 – 5 × 105 cells per antibody	Bulk	Yes	O’Geen et al., 2011
	CUT&RUN	Recombinant protein A-micrococcal nuclease fusion recruited to the antibody targeting chromatin protein of interest; DNA fragments near antibody sites are cleaved, released, and sequenced	5 × 105 cells	Bulk	Yes	Hainer and Fazzio, 2019
	CUT&Tag	A-Tn5 transposase fusion protein bound to antibody; transposase generates fragment libraries for sequencing	100,000 – 500,000 cells	Bulk	Yes	Kaya-Okur et al., 2019
	Joint RNA-seq and CUT&Tag (Paired-Tag)	CUT&Tag followed by RNA-seq: profiling of histone modifications and transcripts in single cells; generates maps of chromatin state and transcript in various tissues by cell type	∼10,000 cells per antibody	Single-cell	Yes	Zhu et al., 2021
	HiCHIP	Comprehensive analysis of single-end and paired-end ChIP-seq reads for protein-DNA interactions	106 – 15 × 106 cells	Bulk	Yes	Yan et al., 2014
	RNA-seq	RNA is isolated from sample and converted into cDNA libraries which undergo NGS.	5 × 104 – 5 × 106 cells	Bulk and single-cell	Yes	Wang et al., 2009; Svensson et al., 2018
Imaging	Multicolor IF-based single cell analysis	Using directly labeled histone modification-specific antibodies to monitor histone levels in single cells			No	Hayashi-Takanaka et al., 2020
	Stochastic Optical Reconstruction Microscopy (STORM)	Single fluorophores blink individually and randomly, enabling precise location of photons, eventually forming full images			No	Xu et al., 2018
Mass spectrometry	LC-MS/MS	Allows for quantification of histone modifications and combinations of modifications	106 – 107 cells	single-cell	Yes	Volker-Albert et al., 2018
Flow cytometry	FACS	Cells are prepared accordingly for high-throughput flow cytometry, and gated based on phenotype of interest; allows for investigation of multiple phenotypes in complex samples	105 – 106 cells	single-cell	Yes	Zahedi et al., 2020
Western blot		Protein is isolated from sample, separated by weight and probed for on gel with specific antibodies			No	Egelhofer et al., 2011