Chromatin Immunoprecipitation

This protocol is modified from the Myer's Lab ChIPseq protocol v011014 found here. The original citation for this methodology is:

Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (80- ) 316: 1497–1502, 2007. doi:10.1126/science.1141319

Buffers and Solutions Needed

• 20% Formaldehyde (from 37% formaldehyde Sigma F87750)
• 2.5M Glycine
• PBS (cold)
• Farnham Lysis Buffer (cold)
• RIPA Buffer (cold)
• Dynabeads (Invitrogen cat#)
• PBS with 5 mg/mL BSA and 1x Protease inhibitor (cold)
• [LiCl Wash Buffer]] make fresh (cold)
• TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)
• ChIP Elution Buffer make fresh
• QIAquick PCR Purification Kit

Equipment

• Cool microfuge and swinging bucket centrifuge down to 4C

This protocol involves preparation of the crosslinked DNA, immunoprecipitation of the DNA and analysis by qPCR. It is possible to stop and freeze the samples after each of these steps.

Crosslinking, Lysis and Shearing of DNA

1. Remove culture plates from the incubator and place at room temperature on the bench.

2. Add formaldehyde to a final concentration of 1% directly to the media of adherent cells growing on tissue culture plates, swirl gently, and incubate at room temperature for 10 minutes.

• If using 10cm dishes add 270ul of 37% formaldehyde

3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix.

• If using 10cm dishes add 0.5mL of the 2.5M glycine stock solution

4. Remove media from plates and wash cells with equal volume cold (4°C) 1X PBS.

• 10mL for 10cm dish

5. Aspirate the PBS and add 2.5 ml cold (4°C) Farnham lysis buffer (make sure to add PI).

6. Scrape the cells off the plate with a cell scraper and transfer into 15-ml conical tubes on ice.

7. Pellet cells at 2,000 rpm for 5 minutes at 4°C.

8. Place cells on ice. Carefully remove supernatant and either proceed to sonication step or snap-freeze in liquid nitrogen and store at -80°C or in liquid nitrogen.

9. Resuspend each fresh or frozen pellet (containing 2 x 107 cells) on ice in 1 ml Farnham Lysis Buffer and mix gently by flicking the test tube. Briefly homogenize cells by running the cells through a 18-gauge needle ~10X. Note: This treatment breaks the cells while keeping the nuclei mostly intact.

10. Collect the crude nuclear prep by centrifuging at 2,000 rpm at 4°C for 5 minutes.

11. Resuspend pellet to 1 ml with RIPA Buffer in a 15 mL falcon tube (Do not vortex the tubes and try to avoid bubbles. Bubbles will cause popping and loss of samples during sonication).

12. Using the Sonics VibraCell Sonicator, sonicate each 1.0 ml ChIP sample on ice, in a cold room, at Power Output 5 watts 6 times for 30 seconds each (60% amplitude), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.

• If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each.

13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant and nano drop samples and calculate the amount needed for 25ug of chromatin.

14. Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below.

Immunoprecipitation

Perform all steps in an ice bucket or in the cold room at 4°C.

Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads

1. Add 20 μl re-suspended magnetic bead slurry (for each sample you plan to use with this antibody) to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Mix well, do not vortex.
2. Place the microfuge tubes on the magnet rack and remove supernatants.
3. Resuspend the beads in 1 ml cold PBS/BSA.
4. Repeat Steps b and c 3 times.
5. Add 200 μl PBS/BSA (for each sample you plan to use this antibody with) to beads.
6. Add 1 μg primary antibody. Mix gently by tapping--Do not vortex beads.
7. Gently mix on a rotator platform for at least 2 hours at 4°C.
8. Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
9. Resuspend in 100 μl PBS/BSA (for each sample you plan to use this antibody with), and proceed to Step 2.

Incubate bead-antibody complex with fragmented, cross-linked chromatin

1. Add 100 μl of antibody-coupled beads from above to each 25ug chromatin of preparation (from Sonication protocol) and incubate on a rotator for 1 hour at room temperature, followed by 1 hour at 4°C.
2. Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.
3. Remove and discard supernatant.
4. Wash beads 5 times with 1 mL LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.
5. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.
6. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.

Reverse cross-linking and recover ChIP DNA

1. Incubate beads from above in a 65°C water bath for 1 hour, shake or vortex every 15 minutes to elute the immuno-bound chromatin from the beads.
2. Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.
3. Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery.
4. Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.

Purification of ChIP DNA

1. Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.
2. Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.
3. Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM, empty, and centrifuge the column containing the bound DNA a second time allow it to dry.
4. Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).

Analysis of Immunoprecipitated DNA

• See RT-PCR primer design for ChIP to design primers if analysing by qPCR