Difference between revisions of "Chromatin Immunoprecipitation"
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===Crosslinking, Lysis and Shearing of DNA=== | ===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. | ||
===Immunoprecipitation=== | ===Immunoprecipitation=== | ||
===Analysis of Immunoprecipitated DNA=== | ===Analysis of Immunoprecipitated DNA=== | ||
Revision as of 16:02, 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.
