Editing BioMicroCenter:ChIP (section)

=== Sample Preparation ===
[[Image:BMC_IPprotocol.png|thumb|right|200px|Testing ChIP conditions with IP-Star]]The IP-star requires 5 million cells per IP. Sonicated chromatin must be submitted at 5-7 million cells per 200 uL volume. Antibodies must be submitted at the same time(at least 3 ug of antibody per IP). It should work with many different cell lysis / crosslinking conditions. [[BioMicroCenter:IPstarTesting|'''We have validated''']] the protocol below and strongly recommend using it as a baseline:

  1. Resuspend fixed cells in 10 mL of [[LB1|LB1]] <br>
  2. Rock cells at 4 degrees for 10 minutes <br>
  3. Pellet cells at 4 degrees for 5 minutes at 1350g <br>
  4. Remove supernatant and resuspend cells in 10mL of [[LB2|LB2]] <br>
  5. Rock cells at 4 degrees for 10 minutes <br>
  6. Pellet cells at 4 degrees for 5 minutes at 1350g <br>
  7. Remove supernatant and resuspend cells in a volume of [[LB3|LB3]] where there is 5 million cells/200uL <br>
      '''**Example:''' Resuspend 25 million cells in 1mL of [[LB3|LB3]]'''**'''
  8. Sonicate cells <br>
  9. Pellet debris at 4 degrees for 10 minutes at 20,000g <br>
  10. Submit lysate for ChIP

Sonication conditions vary broadly between different cell types and different sonicators so you will want to optimize your conditions to produce material in the 100-500bp range. We strongly recommend that you not freeze your lysate before submitting to ChIP prep. Therefore, it is critical that you coordinate with BioMicro Center staff members to be sure that space is available on the IP-Star when your cells are ready. For scheduling,  you may email Shmulik Matola at Shmulikm@mit.edu.