Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications

Abstract

INTRODUCTIONIn cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organization and gene expression in eukaryotes. Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immunofluorescence studies on cultured cells have been pivotal in unraveling the roles of histone modifications. However, to address in detail what happens at specific sites in vivo, chromatin immunoprecipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on non-fixed chromatin from animal cells or tissues (fresh or frozen) to analyze histone modifications at specific chromosomal sites. These protocols are suitable only for analyzing histones and their modifications. For other applications, chromatin immunoprecipitation should be performed on cross-linked chromatin.

Cooperative Binding of Transcription Factors Orchestrates Reprogramming

“…Native ChIP-seq was performed for as described in (Wagschal et al, 2007) for all histone modification except H3K79me2 and H3K9me3. Briefly, 50×10 6 Nuclei were isolated from noncrosslinked cells (MEFs, 48h, pre-i #1 and ESC) by incubation in 2 ml of a hypotonic solution (0.3M sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl 2 , 15mM Tris-HCl pH 7.5, 0.5mM DTT, 0.1% NP40, and protease inhibitor cocktail) followed by centrifugation through a sucrose cushion (1.2M sucrose, 60mM KCL, 15mM NaCl, 5mM MgCl 2 , 0.1mM EGTA, 15 mM Tris-HCl pH 7.5, 0.5mM DTT, and protease inhibitor cocktail).…”

Microprocessor, Setx, Xrn2, and Rrp6 Co-operate to Induce Premature Termination of Transcription by RNAPII

“…Following 64 hr incubation, cells were washed and harvested for native ChIP, performed as described previously (Wagschal et al, 2007), or crosslink ChIP, which was performed as described previously (Nakamura et al, 2012) except that chromatin was precleared at 4°C for 2 hr with 20 μl protein A or G magnetic beads (Invitrogen) that had been preblocked with 1% BSA. Antibodies (2 μg) were incubated for 4 hr with 20 ml of blocked magnetic beads before the addition of sonicated chromatin overnight at 4°C.…”

Epigenetics of human T cells during the G0→G1 transition

“…Preparation of nuclei and ChIP were carried out essentially according to Wagschal et al (2007) with anti-H3Ac (H3K9/K14Ac) (Upstate Inc), antiH3K4me3 (b8580), anti-H3K9me2 (ab7312 or 1220-25, Abcam), and anti-H3K9me3 (Upstate Inc). Extraction of DNA and RNA and bisulfite sequencing were as described previously (Smith et al 2008), and MeDIP was carried out essentially according to Weber et al (2005).…”