Chromatin Immunoprecipitation and Re-ChIP Protocol

Soluble chromatin was precipitated with anti-MUC1-C (#HM-1630-P1ABX; Thermo Fisher Scientific), anti-MTA1 (#5647), anti-MBD3 (#14540), anti-CHD4 (#11912), anti-HDAC1 (#5356; Cell Signaling Technology), anti-MYC (#ab56), anti-H3K27ac (#ab4729; Abcam) or a control non-immune IgG (Santa Cruz Biotechnology). For re-ChIP analysis, anti-MUC1-C complexes from the primary ChIP were eluted and reprecipitated with anti-MYC. The precipitates were 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 compared to IgG (9 (link)). Primers used for ChIP qPCR are listed in Supplemental Table S2.

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Hata T., Rajabi H., Takahashi H., Yasumizu Y., Li W., Jin C., Long M.D., Hu Q., Liu S., Fushimi A., Yamashita N., Kui L., Hong D., Yamamoto M., Miyo M., Hiraki M., Maeda T., Suzuki Y., Samur M.K, & Kufe D. (2019). MUC1-C ACTIVATES THE NuRD COMPLEX TO DRIVE DEDIFFERENTIATION OF TRIPLE-NEGATIVE BREAST CANCER CELLS. Cancer research, 79(22), 5711-5722.

Publication 2019

anti-MUC1-C anti-MTA1 anti-MBD3 anti-CHD4 anti-HDAC1 anti-MYC anti-H3K27ac control non-immune IgG Power SYBR Green PCR Master Mix ABI Prism 7300 sequence detector

Chd4 Chip Chromatin Mbd3 Mta1 Muc1 Primers Prism Sybr green

Corresponding Organization : Dana-Farber Cancer Institute

Other organizations : Roswell Park Comprehensive Cancer Center

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Variable analysis

independent variables

Anti-MUC1-C (#HM-1630-P1ABX; Thermo Fisher Scientific)
Anti-MTA1 (#5647)
Anti-MBD3 (#14540)
Anti-CHD4 (#11912)
Anti-HDAC1 (#5356; Cell Signaling Technology)
Anti-MYC (#ab56)
Anti-H3K27ac (#ab4729; Abcam)
Control non-immune IgG (Santa Cruz Biotechnology)

dependent variables

Soluble chromatin precipitation
Relative-fold enrichment compared to IgG

control variables

Control non-immune IgG (Santa Cruz Biotechnology)

controls

Anti-MUC1-C (#HM-1630-P1ABX; Thermo Fisher Scientific)
Control non-immune IgG (Santa Cruz Biotechnology)

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