Yeast cells By4741 were grown to mid-log on YPD and At “time zero” cells were treated with 1.5mM diamide (D3648, Sigma).
Extracted molecule
genomic DNA
Extraction protocol
At t = 0, 4, 8, 15, 30, and 60 minutes, cells were fixed by 1% formaldehyde, followed after 15 minutes by quenching with 125 mM glycine. Cells were then pelleted, washed with water, and subjected to MNase digestion as previously described (Kaplan et al., Plos Genetics 2008; Radman-Livaja et al., PLoS Genetics, 2010) and immunoprecipitation.
Label
Cy5
Label protocol
ChIP material was amplified using the DNA linear amplification method described previously (Liu et al., 2005; Liu et al., 2003). 3ug of the amplified ChIP products was labeled via the amino-allyl methods as described on http://www.microarrays.org.
Yeast cells By4741 were grown to mid-log on YPD and At “time zero” cells were treated with 1.5mM diamide (D3648, Sigma).
Extracted molecule
genomic DNA
Extraction protocol
At t = 0, 4, 8, 15, 30, and 60 minutes, cells were fixed by 1% formaldehyde, followed after 15 minutes by quenching with 125 mM glycine. Cells were then pelleted, washed with water, and subjected to MNase digestion as previously described (Kaplan et al., Plos Genetics 2008; Radman-Livaja et al., PLoS Genetics, 2010) and immunoprecipitation.
Label
Cy3
Label protocol
ChIP material was amplified using the DNA linear amplification method described previously (Liu et al., 2005; Liu et al., 2003). 3ug of the amplified ChIP products was labeled via the amino-allyl methods as described on http://www.microarrays.org.
Hybridization protocol
Labeled probes (a mixture of Cy5-labeled input and Cy3-labeled ChIP-ed material) were hybridized onto an Agilent yeast tiled oligonucleotide microarray (G4495A) at 65°C for 16 h and washed as described on http://www.microarrays.org
Scan protocol
The arrays were scanned at 5 μ resolution with an Agilent scanner. Images were quantified using Agilent Feature Extraction Software.
Description
MNase-ChIP vs input
Data processing
Agilent Feature Extraction Software was used for background subtraction and LOWESS normalization.
