Chromatin Immunoprecipitation (ChIP) Assay

Preparation of soluble chromatin was carried out as previously described ^{6 (link)}. For HDAC1 and HDAC2 ChIPs the T cells were pre-treated with the crosslinker disuccinimidyl glutarate (2mM mM, AppliChem) for 25 min at room temperature. Chromatin was sonicated with the Bioruptor® Sonication System (Diagenode). For ChIP assays equal amounts of sonicated chromatin were diluted 10-fold and precipitated overnight with the following antibodies: rabbit anti-mouse HDAC1 (affinity purified polyclonal serum ³⁸), mouse anti-mouse HDAC2 (Abcam ab12169), anti-H3K9ac (Millipore 07-449), C-terminal H3 (Abcam ab1791), rabbit IgG (Invitrogen), mouse IgM (Invitrogen) and CBFβ ^{26 (link)} as a control. Chromatin/antibody complexes were isolated using Protein A or Protein G magnetic beads (Dynabeads, Invitrogen). The extracted DNA was used for qRTPCR analysis with an iCycler IQ system (Bio-Rad) and KAPA SYBR FAST qPCR MasterMix (Peqlab). In parallel, PCR reactions with 1:20 dilutions of genomic DNA (input) were carried out. Values for histone H3 modification marks were corrected to the H3 values obtained with C-terminal H3 antibody.

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Boucheron N., Tschismarov R., Goeschl L., Moser M.A., Lagger S., Sakaguchi S., Winter M., Lenz F., Vitko D., Breitwieser F.P., Müller L., Hassan H., Bennett K.L., Colinge J., Schreiner W., Egawa T., Taniuchi I., Matthias P., Seiser C, & Ellmeier W. (2014). CD4+ T cell lineage integrity is controlled by the histone deacetylases HDAC1 and HDAC2. Nature immunology, 15(5), 439-448.

Publication 2014

disuccinimidyl glutarate Bioruptor® Sonication System anti-H3K9ac C-terminal H3 Dynabeads iCycler IQ system KAPA SYBR FAST qPCR MasterMix

Antibodies Antibody Chip assays Chips Chromatin Dilutions Disuccinimidyl glutarate Genomic Histone h3 Mouse Protein a Protein g Rabbit Serum T cells

Corresponding Organization :

Other organizations : Medical University of Vienna, Wellcome Centre for Cell Biology, University of Edinburgh, Max Perutz Labs, Vienna Biocenter, CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences, Washington University in St. Louis, RIKEN Center for Integrative Medical Sciences, Novartis (Switzerland), University of Basel, Friedrich Miescher Institute

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Variable analysis

independent variables

Treatment of T cells with the crosslinker disuccinimidyl glutarate (2mM, AppliChem) for 25 min at room temperature

dependent variables

Levels of HDAC1 and HDAC2 binding to chromatin (measured by ChIP-qPCR)
Levels of H3K9ac and total H3 (measured by ChIP-qPCR)

control variables

Preparation of soluble chromatin (as previously described)
Sonication of chromatin using the Bioruptor® Sonication System (Diagenode)
Equal amounts of sonicated chromatin used for ChIP assays
Precipitation of chromatin/antibody complexes using Protein A or Protein G magnetic beads
QPCR analysis of extracted DNA using an iCycler IQ system (Bio-Rad) and KAPA SYBR FAST qPCR MasterMix (Peqlab)
Parallel PCR reactions with 1:20 dilutions of genomic DNA (input)

positive controls

Rabbit anti-mouse HDAC1 (affinity purified polyclonal serum)
Mouse anti-mouse HDAC2 (Abcam ab12169)
Anti-H3K9ac (Millipore 07-449)
C-terminal H3 (Abcam ab1791)

negative controls

Rabbit IgG (Invitrogen)
Mouse IgM (Invitrogen)
CBFβ

Annotations

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