Brd4 Knockdown and Chromatin Profiling

Brd4 siRNA (Hs_BRD4_6 FlexiTube, SI03190845; Qiagen) or control siRNA (AllStars negative control, 1027281; Qiagen) was transfected into 20861 cells using RNAimax, following the manufacturer’s instruction. The following antibodies were used: ant-γ-H2AX (phospho-histone H2AX [Ser139], 05-636 [Millipore]), anti-Rad51 (ab213 [Abcam]), anti-MED1 (A300-793A [Bethyl Laboratories]), anti-H3K27ac (07-360 [Millipore]), anti-CDK9 (sc-8338 [Santa Cruz]), and anti-Brd4 (A301-985A; 1.2 µg per IP [Bethyl Laboratories]). Affinity-purified Brd4 C-terminus-specific anti-Brd4 antiserum (MCB2) has been described previously (12 (link)). Brd4 (8H2 mouse monoclonal antibody) binds the BDII region and is from Cheng-Ming Chiang (UT Southwestern) (46 (link)). For immunofluorescence, antibodies were diluted 1:100. For ChIP, 3.0 µg antibody was used per IP unless stated otherwise.

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Dooley K.E., Warburton A, & McBride A.A. (2016). Tandemly Integrated HPV16 Can Form a Brd4-Dependent Super-Enhancer-Like Element That Drives Transcription of Viral Oncogenes. mBio, 7(5), e01446-16.

Publication 2016

anti-MED1 (A300-793A anti-CDK9 (sc-8338 anti-Brd4 (A301-985A;

A301 Antibodies Antibody Antiserum Brd4 Cdk9 Cells Chip Histone h2ax Immunofluorescence Med1 Monoclonal antibody Mouse Sirna

Corresponding Organization :

Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Variable analysis

independent variables

Brd4 siRNA (Hs_BRD4_6 FlexiTube, SI03190845; Qiagen)
Control siRNA (AllStars negative control, 1027281; Qiagen)

dependent variables

γ-H2AX (phospho-histone H2AX [Ser139])
Rad51
H3K27ac

control variables

Transfection of 20861 cells using RNAimax, following the manufacturer's instruction
Antibody dilutions for immunofluorescence (1:100)
Antibody amounts for ChIP (3.0 µg per IP unless stated otherwise)

positive controls

Affinity-purified Brd4 C-terminus-specific anti-Brd4 antiserum (MCB2)
Brd4 (8H2 mouse monoclonal antibody) that binds the BDII region

negative controls

Control siRNA (AllStars negative control, 1027281; Qiagen)

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