XChIP was performed as described 30 (link). Briefly, 2–4 ×106 hSAECs per 100 mm dish were washed twice with PBS. Protein-protein crosslinking was first performed with Disuccinimidyl glutarate (Pierce), followed by protein-DNA crosslinking with formaldehyde. Equal amounts of sheared chromatin were immuno-precipitated overnight at 4 ºC with 4 µg of the indicated antibodies in ChIP dilution buffer. Immunoprecipitates were collected with 40 µL Dynabeads Protein-A (Novex), washed, and eluted in 250 µl elution buffer for 15 min at room temperature. Samples were de-crosslinked in 0.2 M NaCl at 65 ºC for 4 h. The precipitated DNA was phenol/chloroform-extracted, precipitated with 100% ethanol and air-dried. The following antibodies were used: IRF1 (#8478, Cell Signaling), IRF3 (sc-9082, Santa Cruz), IRF7 (sc-13041, Santa Cruz), RelA (sc-372, Santa Cruz), CtBP (sc-11390, Santa Cruz), EZH2 (17–662, Upstate), H3K27me3 (39155, Active Motif), H3K4me3 (ab8580, Abcam), ZEB1 (sc-25388, Santa Cruz) and ZEB2 (sc-271984, Santa Cruz).
Protocol detail
Cross-Linked Chromatin Immunoprecipitation (XChIP) Protocol
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Antibodies
Buffer
Chip
Chloroform
Chromatin
Dilution
Dish
Disuccinimidyl glutarate
Ethanol
Ezh2
Formaldehyde
H3k4me3
Irf1
Irf3
Irf7
Nacl
Phenol
Protein
Protein a
Protein dna
Rela
Sc 372
Corresponding Organization : The University of Texas Medical Branch at Galveston
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Variable analysis
independent variables
- Antibodies used for immunoprecipitation: IRF1, IRF3, IRF7, RelA, CtBP, EZH2, H3K27me3, H3K4me3, ZEB1, ZEB2
- Precipitated DNA from chromatin immunoprecipitation (ChIP)
- Number of hSAECs per dish (2-4 × 10^6)
- Protein-protein crosslinking with Disuccinimidyl glutarate
- Protein-DNA crosslinking with formaldehyde
- Chromatin shearing
- Immunoprecipitation overnight at 4°C
- Collection of immunoprecipitates with Dynabeads Protein-A
- Washing of immunoprecipitates
- Elution of immunoprecipitates in elution buffer
- De-crosslinking at 65°C for 4 hours
- Phenol/chloroform extraction and ethanol precipitation of DNA
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