ChIP-PCR Analysis of RORC Promoter

ChIP was performed on total thymocytes from WT or HDAC3-cKO using the SimpleChIP Enzymatic Chromatin IP kit, (Cell Signaling, #9002) per manufacturer’s guidelines. Chromatin was incubated with α-acetyl Histone 3 antibody (Millipore) and control antibodies (Cell Signaling, #9002) overnight at 4°C with rotation. Extracted DNA was analyzed by real-time PCR using previously published primer sequences spanning the RORC promoter (26 (link)).

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Philips R.L., Chen M.W., McWilliams D.C., Belmonte P.J., Constans M.M, & Shapiro V.S. (2016). HDAC3 is required for the down-regulation of RORγt during thymocyte positive selection. Journal of immunology (Baltimore, Md. : 1950), 197(2), 541-554.

Publication 2016

SimpleChIP Enzymatic Chromatin IP kit control antibodies

Antibodies Antibody Chip Chromatin Enzymatic Histone Primer Real time pcr Thymocytes

Corresponding Organization : Mayo Clinic in Arizona

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Variable analysis

independent variables

Genotype (WT or HDAC3-cKO)

dependent variables

Acetylation of Histone 3 at the RORC promoter

control variables

Total thymocytes
SimpleChIP Enzymatic Chromatin IP kit from Cell Signaling
Incubation conditions (overnight at 4°C with rotation)

controls

Positive control: α-acetyl Histone 3 antibody
Negative control: Control antibodies (Cell Signaling, #9002)

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