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 smear in the gel. In the following protocol, we will shear chromatin of chronic myeloid leukemia cell line using sonication.

- To begin, fill a 1.5 millimeter tube with 1 milliliter of 37% formaldehyde. Then, fill another 1.5 milliliter tube with 1 milliliter of 1.25 molar glycine and a 5 milliliter tube with 4 milliliters of PBS. Next, after the stimulation of the cells, spin the cells according to the text protocol, and re-suspend the cells in 500 microliters of PBS and place the tube in an ice-filled box. Add 13.5% microliters of 37% formaldehyde to the cells and mix by pipetting up and down.

Then, incubate the suspension at room temperature. After this, add 57 microliters of 1.25 molar glycine to stop the fixation and allow this suspension to incubate at room temperature for five minutes. Immediately following the incubation period, place the cells on ice and centrifuge the cells at 500 times g for five minutes at 4 degrees Celsius. Then aspirate the supernatant. Next, wash the cells twice with 1 milliliter of ice cold PBS at 200 times g for five minutes at 4 degrees Celsius and remove the supernatant. Resuspend the cell pellet in 5 milliliters of lysis buffer-1 by pipetting up and down.

Then, put the samples on ice and incubate them with shaking for 10 minutes. After this, centrifuge the tubes at 500 times g for five minutes at 4 degrees Celsius. Then, use the pipette to carefully remove the supernatant. Homogenize the cells in 5 milliliters of lysis buffer-2 and pipette up and down to mix. Then, incubate the cells on ice for 10 minutes with shaking. After the incubation period, centrifuge the tubes at 500 times g for five minutes at 4 degrees Celsius and carefully remove the supernatant.

Next, re-suspend the cell pellet in shearing buffer-1 with 1X protease inhibitor. Then, incubate the cell suspension on ice for 10 minutes. Transfer 140 microliters of the cell suspension to sonicator tubes, taking care to avoid forming air bubbles. Place the tubes in a focused ultrasonicator at 7 degrees Celsius and shear for ten to seven and a half minutes. Transfer the cell suspension into 1.5 milliliter tubes. Centrifuge the samples at 15,700 times g for 10 minutes at 4 degrees Celsius and collect the supernatant in a new tube.