Chromatin Shearing: A Method to Generate Chromatin Fragments Using Enzymatic Digestion

- Transcription factors are proteins that bind to a specific site on DNA to regulate gene expression. Errors in transcription factors alter this activity, leading to over expression of genes, which causes cancer. To understand the role of transcription factors and gene regulation, we can perform chromatin immunoprecipitation assay or CHIP, and chromatin shearing is an important step in CHIP workflow.

For chromatin shearing, stabilize the DNA-protein complex by adding formaldehyde. Formaldehyde cross-links proteins to the associated DNA sequence by forming covalent bonds. Next, use a suitable lysis buffer to lyse the cells to release chromatin containing the cross-linked DNA-protein complexes and centrifuge. Shear the chromatin using micrococcal nuclease enzyme. This enzyme cleaves the chromatin between the nucleosomes and digests the loose DNA ends. Centrifuge the mixture and collect the supernatant.

Load the supernatant on TBE agarose gel to analyze the appropriate size of DNA fragments. Apply an electric field across the gel, which causes the negatively charged chromatin DNA to move towards the anode. Smaller fragments move further down the gel. The sheared chromatin appears as a