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Chromatin immunoprecipitation (ChIP and Re-ChIP) assays

JL Judit Liaño-Pons
ML M. Carmen Lafita-Navarro
LG Lorena García-Gaipo
CC Carlota Colomer
JR Javier Rodríguez
AK Alex von Kriegsheim
PH Peter J. Hurlin
FO Fabiana Ourique
MD M. Dolores Delgado
AB Anna Bigas
ME M. Lluis Espinosa
JL Javier León
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Total cell extracts were first lysed with a hypotonic buffer (described in the “Nuclear/cytoplasm fractionation” section) for purifying the nuclear compartment. Then, nuclear lysis and chromatin immunoprecipitation (ChIP) were performed essentially as described54. For the Re-ChIP experiments, we carried out an intermediary step of elution with elution buffer plus 10 mM DTT, 1 h at 37 °C. The resulting DNA was incubated with the second antibody. Immunoprecipitated DNA was purified with the QIAquick PCR Purification Kit (Qiagen, Germantown, MD, USA) and analyzed by qPCR. The SYDH ENCODE project was used as a reference for primer designing on the human NFKBIA gene (http://genome.ucsc.edu/ENCODE). The primers and antibodies used are described in Supplementary Tables 2 and 3, respectively.

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