The labeling of DNA samples for ChIP-ChIP analysis was performed by NimbleGen Systems, Inc. Briefly, each DNA sample (1 ug) was denatured in the presence of 5'-Cy3- or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water.
The hybridization of DNA samples for ChIP-ChIP analysis was performed by NimbleGen Systems, Inc. Briefly, 13ug of the Cy5-labeled ChIP sample and 13ug of the Cy3-labeled total sample were mixed, dried down, and resuspended in 40 ul of NimbleGen Hybridization Buffer (NimbleGen Systems) plus 1.5 ug of human COT1 DNA. After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems), dried by centrifugation.
Scan protocol
The arrays were scanned at 5-um resolution using the GenePix 4000B scanner (Axon Instruments).
Description
Normal Liver. Note array 2 of 2 5 kb promoter array; complement is 88819; see Acevedo et al., 2008 Cancer Res in press