Description

ChIP analysis was performed using antibodies to E2F1 and Myc in HeLa cells. E2F1 and Myc protein are transcription factors related to growth. E2F1 is important in controlling cell division, and C-Myc is associated with cell proliferation and neoplastic disease. Three independently crosslinked preparations of HeLa cells were used to provide three independent biological replicates. ChIP assays were performed using the protocol found at Farnham Lab Protocols. Array hybridizations were performed using standard NimbleGen Systems conditions.

Methods

Ratio intensity values (antibody vs. total) for each of three biological replicates were calculated and converted to log₂. Peaks were identified independently for each of the three E2F1 and the three Myc ChIP-chip experiments using the Tamalpais program. The identified peaks from the L1 categories for the three E2F1 or three Myc experiments were then compared. All regions reported here as binding sites were identified in at least two of the three E2F1 or at least two of the three Myc ChIP-chip assays.

Verification

Primers were chosen to correspond to 13 individual peaks. PCR reactions were performed for each of the 13 primer sets using amplicons derived from each of three biological samples (39 reactions). The PCR reactions confirmed that all of the 13 chosen peaks were bound by E2F1 in all three biological samples.

Credits

These data were contributed by Mike Singer, Kyle Munn, Nan Jiang, Todd Richmond and Roland Green of NimbleGen Systems, Inc., and Matt Oberley, David Inman, Mark Bieda, Shally Xu and Peggy Farnham of Farnham Lab.

Reference

Bieda M, Xu X, Singer MA, Green R, Farnham PJ. Unbiased location analysis of E2F1-binding sites suggests a widespread role for E2F1 in the human genome. Genome Res. 2006 May;16(5):595-605.