162
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Changes
→Crosslinking, Lysis and Shearing of DNA
8. Place cells on ice. Carefully remove supernatant and either proceed to sonication step or snap-freeze in liquid nitrogen and store at -80°C or in liquid nitrogen.
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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.
10. Collect the crude nuclear prep by centrifuging at 2,000 rpm at 4oC for 5 minutes.
11. Resuspend pellet to 1 ml with RIPA Buffer.
[[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.
14. Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below.
===Immunoprecipitation===
===Analysis of Immunoprecipitated DNA===