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Histone Modification ChIP-seq on Arabidopsis thaliana Plantlets.

Bio-protocol (2021-12-04)
Juan S Ramirez-Prado, David Latrasse, Moussa Benhamed
ABSTRACT

Characterizing the molecular mechanisms regulating gene expression is crucial for understanding the regulatory processes underlying physiological responses to environmental and developmental signals in eukaryotes. The covalent modification of histones contributes to the compaction levels of chromatin, as well as the recruitment of the transcriptional machinery to specific loci, facilitating metastable changes in gene activity. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) has become the gold standard method for determining histone modification profiles among different organisms, tissues, and genotypes. In the current protocol, we describe a highly robust method for performing ChIP-seq of histone modifications in Arabidopsis thaliana plantlets. Besides its robustness, this method uses in-house-prepared buffers for chromatin extraction, immunoprecipitation, washing, and elusion, making it cost-effective in contrast to commercial kits.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycine, ACS reagent, ≥98.5%
Sigma-Aldrich
Anti-acetyl-Histone H3 (Lys14) Antibody, serum, Upstate®
Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-monomethyl-Histone H3 (Lys27) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-acetyl-Histone H3 (Lys9) Antibody, serum, Upstate®
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt solution, BioUltra, pH 8.0, ~0.5 M in H2O
Roche
cOmplete, EDTA-free Protease Inhibitor Cocktail, Tablets provided in glass vials
Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys27) Antibody, Upstate®, from rabbit