ChromaFlash Chromatin Extraction Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Spin the solution down to the bottom prior to use. - Check if any buffers contain salt precipitates before use. If so, shake the buffer until the salts are re-dissolved.	-For optimal preparation, the input amount should be 1 to 5 x 106 cells or 50 to 200 mg tissues. -Avoid multiple freeze/thaw cycles.

Upstream tips
- Spin the solution down to the bottom prior to use. - Check if any buffers contain salt precipitates before use. If so, shake the buffer until the salts are re-dissolved.
Protocol tips
-For optimal preparation, the input amount should be 1 to 5 x 106 cells or 50 to 200 mg tissues. -Avoid multiple freeze/thaw cycles.

Publication protocol

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to generate extensive genome-wide data sets profiling four selected histone modifications across the four exposure groups. Frozen hearts (3–4 from litter mates) were pooled (~250 mg) and homogenized followed by chromatin isolation using P-2001 ChromaFlashTM Chromatin Extraction Kit (Epigentek, Farmingdale, NY, USA). Chromatin was sheared using Episonic2000 Sonication System (Epigentek, Farmingdale, NY, USA) in 300 μL of ChIP buffer, followed by a quality control of sheared chromatin using fluorescence quantification. A minimum of 4 μL of sheared chromatin was purified to obtain DNA, which was eluted with 20 μL of water. DNA fragment (100–300 bp) was quality-checked using a bioanalyzer. For each ChIP reaction, 10 μg of chromatin, 3 μg of histone 3 trimethylated at lysine 4 (H3K4me3) polyclonal antibody or 3 μg of histone 3 trimethylated at lysine 27 (H3K27me3) antibody or 3 μg of histone acetylated at lysine 9 and 14 (H3K9/14ac) polyclonal ChIP-grade antibody (all antibodies were from Epigentek, Farmingdale, NY, catalogue numbers are: A4021, A4039, A4033) and 6 μL of protein A/G beads were used. ChIPed deoxyribonucleic acid (DNA) was eluted in 20 μL of water. Non-ChIPed DNA was used as input controls for the test groups. Since acetylation is a gene-activating mark irrespective of the associated lysine residue and the same antibody targeted both residues, observed acetylation peaks for lysine 9 and 14 are presented together and are referred to as H3Ac, hereafter. Therefore, a total of 12 biological datasets plus four input control datasets were generated.

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Manufacturer protocol

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