Native Chromatin Immunoprecipitation of Neurosphere Cells

Take neurosphere cells in a buffer containing detergents, calcium, and protease inhibitors. 

The detergent permeabilizes cells, while the inhibitor inactivates proteases to preserve the chromatin-bound target protein.

Add nuclease and incubate. The nuclease uses calcium to digest the chromatin.

Add an EDTA-containing buffer that chelates calcium, inactivating the nuclease.

Add lysis buffer to lyse the cells, releasing the chromatin.

Centrifuge and collect the supernatant.

Add antibodies specific to the target protein and incubate with rotation. Centrifuge and remove the supernatant.

Introduce magnetic beads targeting the antibody and incubate with rotation.

Add lysis buffer to separate non-specifically bound DNA.

Magnetically capture the beads and remove the supernatant.

Add a high-salt buffer to separate non-specifically bound proteins.

Magnetically capture the beads and remove the supernatant.

Add a stabilization buffer, magnetically capture the beads, and remove the contaminants.

Resuspend in a proteinase-K buffer to digest the proteins, releasing the DNA.

Once the neurospheres are harvested, centrifuge 1 times 10 to the 6 neurosphere cells per immunoprecipitation at 300 times g for 5 minutes. After centrifuging, decant the supernatant and re-suspend the neurosphere pellet in one milliliter of balanced salt solution. Transfer the suspension to low protein binding 1.5 milliliter microcentrifuge tubes then centrifuge as before.

Next, decant the supernatant and re-suspend the cell pellet in 95 microliters of digestion buffer supplemented 1 to 100 with protease inhibitor cocktail per 1 times 10 to the 6 cells. Immediately pipette the cells up and down to prevent clumping and avoid creating any bubbles. Then mix 5 microliters of 0.1x micrococcal nuclease and 145 microliters of digestion buffer, and add 5 microliters of diluted micrococcal nuclease per 1 times 10 to the 6 cells. Flick the tube to mix and place the tube in a 37 degrees Celsius heat block for exactly 12 minutes.

The micrococcal nuclease digestion to fragment chromatin is the most critical step. Proper chromatin fragmentation is important for achieving a high resolution for ChIP. To ensure this step is successful, incubate multiple samples one at a time to ensure over digestion does not occur.

After 12 minutes, add 10 microliters of 10x micrococcal nuclease stop buffer per 1 times 10 to the 6 cells. Samples must be kept on ice from this point onwards. Bio-analyzer analysis of the micrococcal nuclease digested sample demonstrates that most of the DNA has been fragmented into mono, di, and tri-nucleosomes. Next, add 110 microliters of 2x RIPA buffer supplemented 1 to 100 with protease inhibitor cocktail per 1 times 10 to the 6 cells.

Flick the tube to mix then centrifuge for 15 minutes at 4 degrees Celsius and 1,700 times g. Transfer the supernatant which contains the chromatin to a regular 1.5 milliliter microcentrifuge tube and place on ice. Use RIPA buffer supplemented with protease inhibitors at 1 to 1,000 to dilute the chromatin so that each IP has a volume of 100 to 200 microliters. Then remove an aliquot equivalent to 10% of the ChIP volume as the input sample.

Prepare the input by adding 100 microliters of TE buffer supplemented with proteinase-K and incubate at 55 degrees Celsius for one hour. After purifying the input with a PCR purification kit, measure the concentration of the input using a micro volume spectrophotometer. Record the results in a notebook. Add 10 microliters of washed protein A and G magnetic beads per IP that will be performed on each sample and incubate for one hour at 4 degrees Celsius with rotation at 20 RPM.

Place the sample on a magnetic holder and allow the beads to separate. Then transfer the volume required into new 1.5 milliliter tubes. Add the desired antibody to each tube, and then wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 degrees Celsius with rotation as before. The next day, spin the tubes at 2000 times g for 10 seconds. Then add 10 microliters of beads to each IP and incubate for three hours at 4 degrees Celsius with rotation at 20 RPM.

Then place the samples on a magnetic stand and allow the magnetic beads to separate. Use a pipette to remove the supernatant. Then add 150 microliters of RIPA buffer with protease inhibitors to each IP and incubate at 4 degrees Celsius at 20 RPM for 5 minutes. After the incubation period, place the samples on a magnetic stand and allow the magnetic beads to separate. Use a pipette to remove the supernatant. Then add 150 microliters of lithium chloride buffer supplemented with protease inhibitors at low concentration to each IP and incubate at 4 degrees Celsius with rotation at 20 RPM for 5 minutes.

After the incubation period, place the samples on a magnetic stand and allow the magnetic beads to separate. Then use a pipette to remove the supernatant. Then add 150 microliters of cold TE without protease inhibitors to the beads and incubate at 4 degrees Celsius with rotation at 20 RPM for 5 minutes. After the final washing step, re-suspend sample in 100 microliters of TE buffer supplemented with the final concentration of 0.5 milligrams per millilitre proteinase-K and incubate at 55 degrees Celsius for one hour.