Difference between revisions of "Chromatin Immunoprecipitation"

Revision as of 16:16, 20 January 2016

This protocol is modified from the Myer's Lab ChIPseq protocol v011014 found here. The original citation for this methodology is:

Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (80- ) 316: 1497–1502, 2007. doi:10.1126/science.1141319

Before You Start

Buffers and Solutions Needed

• 20% Formaldehyde (from 37% formaldehyde Sigma F87750)
• 2.5M Glycine
• PBS (cold)
• Farnham Lysis Buffer (cold)
• RIPA Buffer (cold)
• Dynabeads (Invitrogen cat#)
• PBS with 5 mg/mL BSA (cold)
• [LiCl Wash Buffer]] (cold)
• TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)
• ChIP Elution Buffer

Equipment

• Cool microfuge and swinging bucket centrifuge down to 4C

Protocol

This protocol involves preparation of the crosslinked DNA, immunoprecipitation of the DNA and analysis by qPCR. It is possible to stop and freeze the samples after each of these steps.

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.

3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix.

4. Remove media from plates and wash cells with equal volume cold (4°C) 1X PBS.

5. Aspirate the PBS and add 5-8 ml cold (4°C) Farnham lysis buffer.

6. Scrape the cells off the plate with a cell scraper and transfer into 15-ml conical tubes on ice.

7. Pellet cells at 2,000 rpm for 5 minutes at 4°C.

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.

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