Formaldehyde cross-linked and sheered soluble chromatin was precipitated with pre-cleared magnetic dynabeads (ThermoFisher Scientific) and 2 μg/ml of anti-MUC1-C (HM-1630-P1ABX; ThermoFisher Scientific), anti-E2F1 (3742; CST), anti-STAT3 (9139; CST), anti-BRG1 (ab110641; abcam), anti-ARID1A (12354, 2 μg/ml; CST) or a control non-immune IgG (Santa Cruz Biotechnology). The DNA-antibody precipitates were reverse cross-linked at 65°C for 18 h. DNAs were purified using gel extraction columns (QIAGEN, Germantown, MD, USA) and analyzed by qPCR using the Power SYBR Green PCR Master Mix and the ABI Prism 7300 sequence detector (Applied Biosystems). Data are reported as relative fold enrichment (10 (link)). Primers used for ChIP qPCR are listed in Supplementary Table 2 .
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Chromatin Immunoprecipitation Assay Protocol
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Other organizations : Dana-Farber Cancer Institute, Harvard University, Roswell Park Comprehensive Cancer Center, King Khalid University Hospital, King Saud University, Keio University
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Variable analysis
independent variables
- Antibody used for chromatin immunoprecipitation (ChIP)
- Concentration of antibodies used for ChIP (2 μg/ml)
- Relative fold enrichment of DNA fragments precipitated by the different antibodies
- Formaldehyde cross-linked and sheered soluble chromatin
- Pre-cleared magnetic dynabeads (ThermoFisher Scientific)
- Reverse cross-linking at 65°C for 18 h
- DNA purification using gel extraction columns (QIAGEN)
- QPCR analysis using Power SYBR Green PCR Master Mix and ABI Prism 7300 sequence detector (Applied Biosystems)
- Positive control: None mentioned
- Negative control: Control non-immune IgG (Santa Cruz Biotechnology)
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