''Perform all steps in an ice bucket or in the cold room at 4°C.''
1. ==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads:==== <nowiki> a. # Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Vortex briefly to mix well. b. # Place the microfuge tubes on the magnet rack and remove supernatants. c. # Resuspend the beads in 1 ml cold PBS/BSA. d. # Repeat Steps b and c 3 times. e. # Add 200 μl PBS/BSA to beads. f. # Add 5 μg primary antibody. Do not vortex beads after adding the antibody. g. # Gently mix on a rotator platform for at least 2 hours at 4°C. h. # Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash. i. # Resuspend in 100 μl PBS/BSA, and proceed to Step 2.</nowiki>
2. ==== Incubate bead-antibody complex with fragmented, cross-linked chromatin==== <nowiki> a. # Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at 4°C. b. # Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack. c. # Remove and discard supernatant. d. # Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator. e. # 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. f. # Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.</nowiki>
3. ==== Reverse cross-linking and recover ChIP DNA ==== <nowiki> a. # Incubate bead pellet from step 2.f 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. b. # Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes. c. # Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery. d. # Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.</nowiki>
===Analysis of Immunoprecipitated DNA===