Chromatin Immunoprecipitation (ChIP) Assay

NSCs were expanded to approximately 8 million cells, of which half were enriched for NRF2 using siRNA knockdown of NRF2’s negative regulator KEAP1 and the other half were transfected with a nontargeting siRNA control. Samples were prepared following the SimpleChIP® Plus Enzymatic Chromatin IP Kit protocol supplied by the manufacturer (Cell Signaling Technology). Briefly, 48 hr post-transfection, NRF2 was cross-linked to DNA using 1.5% formaldehyde. Nuclei were collected and lysed by sonication. Chromatin DNA was digested with micrococcal nuclease for 18 min into small fragments (150–900 bp). Nuclear extracts were incubated overnight with NRF2 antibody (cat. no. 12721; Cell Signaling Technology) and antibody-bound complexes were captured by SureBeads™ protein G magnetic beads (Bio-Rad). Bound DNA was purified and underwent quantitation by PCR using primers for putative SPP1 ARE, NQO1 ARE (Chorley et al. 2012) and RPL30-exon 3 (Cell Signaling Technology).