ChIP was conducted as previously described18 (link). For ChIP-qPCR experiments (see below) a 10 min crosslinking time was used for both the FLAG M1 antibody (overexpression experiments) and SETX antibody. Sonication was performed in a refrigerated Bioruptor (Diagenode) and conditions were optimized to generate DNA fragments of approximately 200–1000 bp. Lysates were pre-cleared with the appropriate isotype control for 3 hours. Specific antibodies (Ab) were coupled with anti-mouse- (Dynabeads® M-280 Sheep Anti-Mouse IgG, Invitrogen 112-02), or anti-rabbit IgG bound magnetic beads (Dynabeads® M-280 Sheep Anti-Mouse IgG, Invitrogen, 112-04) for 6 hours. Antibody bound beads and chromatin were then immunoprecipitated at 4°C rotating overnight. After the wash steps, reverse crosslinking was carried out at 65°C overnight. ChIP DNA was then isolated after RNase digestion and proteinase K digestion, using the QIAGEN MinElute kit (QIAGEN, 28004) and used for downstream applications. Statistical significance of ChIP qPCR analysis was determined using two-tailed Student’s paired t-test.
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Chromatin Immunoprecipitation (ChIP) Protocol
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Corresponding Organization : Icahn School of Medicine at Mount Sinai
Other organizations : Institute of Molecular and Cell Biology, McGill University, Salk Institute for Biological Studies, Roche (Switzerland), University of Queensland
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Variable analysis
independent variables
- Crosslinking time (10 minutes)
- Antibody used (FLAG M1 antibody for overexpression experiments, SETX antibody)
- DNA fragment size (approximately 200–1000 bp)
- Sonication conditions (optimized to generate DNA fragments of approximately 200–1000 bp)
- Lysate pre-clearing (with appropriate isotype control for 3 hours)
- Antibody coupling (with anti-mouse or anti-rabbit IgG bound magnetic beads for 6 hours)
- Immunoprecipitation (at 4°C rotating overnight)
- Wash steps
- Reverse crosslinking (at 65°C overnight)
- DNA isolation (RNase digestion and proteinase K digestion, using the QIAGEN MinElute kit)
- Positive control: Not specified
- Negative control: Appropriate isotype control for 3 hours
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