We genetically engineered S. cerevisiae to contain a cysteine at position 47 in histone H4. Cells grown to mid-log phase were harvested, permeabilized and labeled with N(1,10 phenanthroline- 5-yl) iodoacetamide. The label covalently bound to the cysteine and allowed for copper chelation. Copper chloride, mercaptoproprionic acid and hydrogen peroxide were added sequentially creating hydroxyl radicals that cleaved the nucleosomal DNA at sites flanking the center. After the mapping reaction, the genomic DNA was purified from the cells and ran on an agarose gel. The shortest molecular weight DNA fragment (~150-200bp) was purified and prepared for highthroughput parallel sequencing.
Protocol detail
Histone H4 cysteine mapping
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Acid
Agarose
Cells
Chloride
Copper
Cysteine
Genomic
Histone h4
Hydrogen peroxide
Hydroxyl radicals
Iodoacetamide
Nucleosomal
Phenanthroline
Corresponding Organization :
Other organizations : Northwestern University
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Variable analysis
independent variables
- Genetically engineered S. cerevisiae to contain a cysteine at position 47 in histone H4
- Copper chelation at the cysteine residue
- Hydroxyl radical cleavage of nucleosomal DNA at sites flanking the center
- Length of DNA fragments (~150-200bp)
- Cells grown to mid-log phase
- Permeabilized cells
- Labeling with N(1,10 phenanthroline- 5-yl) iodoacetamide
- Addition of copper chloride, mercaptoproprionic acid, and hydrogen peroxide sequentially
- Purification of genomic DNA
- Agarose gel electrophoresis
- Purification of the shortest DNA fragment (~150-200bp)
- Preparation for high-throughput parallel sequencing
- Not explicitly mentioned
- Not explicitly mentioned
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