Natural variation of histone modification and its impact on gene expression in rat
Natural variation of histone modification and its impact on gene expression in rat
Rats were kept in the animal facility of the Institute of Physiology, Czech Academy of Science (Prague, Czech Republic) in a climate-controlled environment with 12h light/dark cycles. Rats were housed in polystyrene cages containing wood shavings and fed standard rodent chow and water ad libitum. Six weeks old male rats were sacrificed by cervical dislocation and tissue (left ventricle of the heart and liver) was collected between 8 and 10 am, snap frozen in liquid nitrogen and stored at -80 C. All animal procedures were in accordance with the Animal Protection Law of the Czech Republic. ChIP samples were prepared as previously described (Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823-37, 2007) with following changes: Frozen LV and liver tissue was crunched using pestle and mortar. 125mg tissue was homogenized in a Potter- Elvehjem tissue grinder, nuclei were isolated and digested with MNase (Sigma- Aldrich) to generate native chromatin templates consisting mainly of mononucleosomes. Chromatin was precipitated with anti-H3K4me3 (NEB, 9751 S), anti-H3K27me3 (Millipore, 07-449), anti-H3K4me1 (Abcam, ab8895), or anti- H4K20me1 (Abcam, ab9051) and antibody-bound DNA fragments were purified by MinElute PCR Purification Kit (QIAGEN). Specificity of immunoprecipitation was confirmed with known genes by qPCR. 50ng of DNA was used for construction of ChIP-seq libraries according to the manufacturer's protocol (Illumina/Solexa).
Experimental Factor: Genotype
anti-H3K4me3
Experimental Factor: OrganismPart
heart
Experimental Factor: StrainOrLine
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