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 (this is around 60% amplitude-but it 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.Dilute to 500-1.5mL cold 1x TE (this is based on the amount of lysis buffer you used).Resuspend fixed cells in 50-150uL lysis buffer(depending on amount of tissue) and incubate for 10 min on ice.Store cells at -80C or proceed to sonication.Centrifuge at low speed (10 min at 2500 x g/RCF).Decant the supernatant and was once with 5mL cold 1x PBS.

Centrifuge samples at low speed (15 min at 2000 x g/RCF).Stop the crosslinking reaction by adding 0.275mL of 2.5 M glycine to a final concentration of 0.125 M.(Add this to all samples at the same time) Add 0.5mL cross linking buffer and rotate the tube at room temperature for 20 min.Add cold 1x PBS up to 5mL to all tubes as you go.Transfer tissue into a 15mL conical tube using a clean spatula or pipette.Clean the mortar and pestle with 10% bleach and 70% ethanol between samples.Use a clean, cold spatula to scoop the ground tissue back to the original tube.Grind up the sample with liquid nitrogen using the mortar and pestle.Remove tissue from tube and place into liquid nitrogen with a clean, cold spatula.Pour Liquid nitrogen into a mortar and pestle.

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

Chip seq from flashfrozen cells