Chromatin Immunoprecipitation Protocol

ChIP assays were performed as described by using EZ-Magna ChIP (17-10086, Millipore).(51 (link),63 (link)) Sonicated extracts were precleared and incubated with Abs specific to Ezh2 (39901, active motif), H3K27me3 (PAb-069-050, Diagenode) or H3K4me3 (9751S, Cell Signaling) at 4°C overnight on a 360°C rotator. The immunoprecipitated DNA was quantitated by real-time quantitative PCR. The primer sequences are listed in Supplemental Table 2.

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Tong Q., He S., Xie F., Mochizuki K., Liu Y., Mochizuki I., Meng L., Sun H., Zhang Y., Guo Y., Hexner E, & Zhang Y. (2014). Ezh2 regulates transcriptional and post-translational expression of T-bet and promotes Th1 cell responses mediating aplastic anemia in mice. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5012-5022.

Publication 2014

EZ-Magna ChIP PAb-069-050

Chip Chip assays Ezh2 H3k4me3 Primer Quantitative real time pcr

Corresponding Organization : Temple University

Other organizations : Second Military Medical University, Shanghai Institutes for Biological Sciences, University of Pennsylvania

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Variable analysis

independent variables

Antibodies used for immunoprecipitation (Ezh2, H3K27me3, H3K4me3)

dependent variables

Immunoprecipitated DNA quantified by real-time quantitative PCR

control variables

Sonicated extracts precleared prior to immunoprecipitation
Immunoprecipitation performed at 4°C overnight on a rotator

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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