Chromatin Immunoprecipitation Analysis Protocol

ChIP assays were performed as described previously (41 (link)). Sheared chromatin from O3-73 and its derivatives were prepared after fixation with 1% formaldehyde and sonication with Diagenode Bioruptor Pico for 20 cycles (30 s each, vortexed every 5 cycles). ChIP antibodies of rabbit IgG isotypes were coupled to Protein A Dynabeads and were obtained from the following sources: RUNX1 C terminus was provided by Dr. Takeshi Egawa (42 (link)), ETS-1 C20 (Santa Cruz sc-350×), normal rabbit IgG (Santa Cruz sc-2027×), Gal4DBD (Santa Cruz sc-577×), total Histone H3 (Abcam ab1791), H3K4Me3 (Abcam ab8580) and H3K27Ac (Abcam ab4729). ChIP antibodies of mouse IgG isotypes were coupled to Protein G Dynabeads and were obtained from the following sources: p300 (Santa Cruz sc-584×) and normal mouse IgG (Santa Cruze sc-2025). Eluted ChIP and input DNAs were purified with Qiagen QIAquick PCR purification kit and analyzed by qPCR using primers and conditions provided in Table S1.

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Zhao J.Y., Osipovich O., Koues O.I., Majumder K, & Oltz E.M. (2017). Activation of Mouse Tcrb: Uncoupling RUNX1 Function from its Cooperative Binding with ETS1. Journal of immunology (Baltimore, Md. : 1950), 199(3), 1131-1141.

Publication 2017

Bioruptor Pico ETS-1 C20 normal rabbit IgG Gal4DBD total Histone H3 H3K4Me3 H3K27Ac normal mouse IgG QIAquick PCR purification kit

Chip Chip assays Chromatin Derivatives Dnas Formaldehyde H3k4me3 Histone h3 Igg antibodies Isotypes Mouse P300 Primers Protein a Protein g Rabbit Runx1

Corresponding Organization : Washington University in St. Louis

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Variable analysis

independent variables

Sheared chromatin from O3-73 and its derivatives

dependent variables

ChIP-qPCR analysis

control variables

Fixation with 1% formaldehyde
Sonication with Diagenode Bioruptor Pico for 20 cycles (30 s each, vortexed every 5 cycles)
Antibodies coupled to Protein A Dynabeads and Protein G Dynabeads

positive controls

RUNX1 C terminus antibody provided by Dr. Takeshi Egawa
ETS-1 C20 antibody (Santa Cruz sc-350×)
Gal4DBD antibody (Santa Cruz sc-577×)
Total Histone H3 antibody (Abcam ab1791)
H3K4Me3 antibody (Abcam ab8580)
H3K27Ac antibody (Abcam ab4729)
P300 antibody (Santa Cruz sc-584×)

negative controls

Normal rabbit IgG (Santa Cruz sc-2027×)
Normal mouse IgG (Santa Cruz sc-2025)

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