The tissue fixation portion of this protocol was taken from the Ren Lab ENCODE protocol for brown adipose tissue and can be found here [1].

Buffers and Solutions Needed

Mass of tissue needed: TBD Crosslinking Buffer Lysis Buffer TE 2.5 M Glycine Cold 1x PBS ChIP Elution Buffer (make fresh) LiCl Wash Buffer

Pulverization of Tissue

*Note: Ensure the samples are kept frozen on dry ice throughout pulverization.

1. Pour Liquid nitrogen into a mortar and pestle.
2. Remove tissue from tube and place into liquid nitrogen with a clean, cold spatula
3. Grind up the sample with liquid nitrogen using the mortar and pestle.
4. Use a clean, cold spatula to scoop the ground tissue back to the original tube.
5. Place the sample back on dry ice.
6. Clean the mortar and pestle with 10% bleach and 70% ethanol between samples

You can continue or store at -80C.

Cross-linking of Tissue

1. Transfer tissue into a 15mL conical tube using a clean spatula or pipette
2. Add cold 1x PBS up to 5mL to all tubes as you go.
3. Add 0.5mL cross linking buffer and rotate the tube at room temperature for 20 min. (Add this to all samples at the same time)
4. Stop the crosslinking reaction by adding 0.275mL of 2.5 M glycine to a final concentration of 0.125 M.
5. Rotate at room temperature for 5 min.
6. Centrifuge samples at low speed (15 min at 2000 x g/RCF).
7. Decant the supernatant and was once with 5mL cold 1x PBS.
8. Centrifuge at low speed (10 min at 2500 x g/RCF).
9. Decant the supernatant.
10. Store cells at -80C or proceed to sonication.

Sonication of Tissue

1. Resuspend fixed cells in 50-150uL lysis buffer(depending on amount of tissue) and incubate for 10 min on ice.
2. Dilute to 500-1.5mL cold 1x TE (this is based on the amount of lysis buffer you used).
3. 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--this ranges depending on how much tissue so play around with amplitude for your sample to get near 5W), 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.

1. 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.

1. 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. Note: For each wash, you will want to invert the tube while on the magnet rack to collect the beads that remain in the liquid at the top of the tube. This will allow you aspirate the liquid from the top of the tube between washes without sucking up any of the beads.
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. Turn water bath to 65°C before next step.
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.
4. Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.
5. 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