normalization data transformation protocol
ChIP-Chip data was normalized as described by Lavoie et al. (2010) PLoS Biol 8(3): e1000329. ID_REF = VALUE = Normalized log10 ratio (ChIP/Input)
array scanning protocol
Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 10-µm resolution.
The microarray slides were pre-hybridized for 1 hour at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. The slides were washed twice for 10 min at 42°C with 1x SSC, 0.1% SDS, twice for 10 min at 37°C with 0.1x SSC, 0.1% SDS, and finally, with quick consecutive washes in three 0.1x SSC baths. Slides were air-dried.
Indirect labeling with Cy3 and Cy5 was performed as described by Lavoie et al. BMC Genomics 2008, 9:578. The labeled DNA was purified with QIAquick PCR Purification Kit (Qiagen).
sample treatment protocol
For ChIP-chip analysis, an overnight cultures in YPD were diluted to OD600nm =0.2 and grown to OD600nm=2 in 40 ml YPD at 30oC.
nucleic acid extraction protocol
ChIP-chip experiment was performed as described by Lavoie et al. BMC Genomics 2008, 9:578. RNAs were extracted as described by Sellam et al. (2009) Eukaryotic Cell 8 (8), 1174-1183, using the RNeasy Mini kit (Qiagen).