Chromatin Immunoprecipitation of Transcription Factors

A total of 1 x 10⁷ BM-Mϕ were treated with a vehicle control or cytokine for 24 hours and ChIP was performed as previously described [24 (link)]. DNA was fragmented by sonication using a Branson Sonifier 450 (Branson Ultrasonics). For immunoprecipitation, the following antibodies were used: IRF7 (Santa Cruz Biotechnology), STAT1 (Abcam), H3K9me3 (Abcam), Setdb2 (Dr. Yali Dou, University of Michigan), and rabbit polyclonal IgG (Millipore). DNA was assessed by RT-PCR using custom primers (S2 Table).

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Kroetz D.N., Allen R.M., Schaller M.A., Cavallaro C., Ito T, & Kunkel S.L. (2015). Type I Interferon Induced Epigenetic Regulation of Macrophages Suppresses Innate and Adaptive Immunity in Acute Respiratory Viral Infection. PLoS Pathogens, 11(12), e1005338.

Publication 2015

Branson Sonifier 450 STAT1 H3K9me3 Setdb2 rabbit polyclonal IgG

Antibodies Chip Cytokine Immunoprecipitation Irf7 Primers Rabbit Rt pcr Stat1 Ultrasonics

Corresponding Organization : Nara Medical University

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Variable analysis

independent variables

Treatment with vehicle control or cytokine

dependent variables

DNA binding of IRF7, STAT1, H3K9me3, and Setdb2 as measured by ChIP-qPCR

control variables

BM-Mϕ cell line
Sonication protocol for DNA fragmentation
Antibodies used for immunoprecipitation (IRF7, STAT1, H3K9me3, Setdb2, and rabbit polyclonal IgG)

controls

Positive control: Rabbit polyclonal IgG antibody
Negative control: Not explicitly mentioned

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