NFATC1 ChIP-seq with polyclonal antibody
To induce nuclear localization of NFATC1 Jurkat cells were exposed to 2uM ionomycin and 100ng/ml PMA for twenty-four hours. Proteins were cross-linked to DNA by incubation of cells for 10 min in medium containing 1% formaldehyde at room temperature, after which the cross-linking was quenched, nuclei extracted and DNA fragmented by sonication. Precleared samples were incubated with 4ug of specific antibody overnight at 4C and the antibodies collected by incubation with 30 ul of protein G- Sepharose beads for 2-3 h at  4C followed by centrifugation at 800 x g. Subsequently the beads were washed and precipitated chromatin complexes were eluted, and the cross-links reversed by incubating overnight at 65C. DNA was extracted with QiaQuick PCR purification kit. Illumina sequencing library was prepared with standard methods. See Jolma et al. 2009 for details.