162
edits
Changes
→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 0.5mL of the 2.5M glycerol stock solution).
3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix.
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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.''
[[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.
13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant.