|
Status |
Public on Jan 28, 2009 |
Title |
ChIP Input cell cycle CCA, 40 min | array na_13_079 |
Sample type |
genomic |
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|
Channel 1 |
Source Name |
Amplified sonicated DNA
|
Organism(s) |
Saccharomyces cerevisiae |
Characteristics |
Strain: BY4741 bar1(del)
|
Extracted molecule |
genomic DNA |
Extraction protocol |
DNA was isolated from mononucleosomal cell extract via proteinase K digestion and phenol:chloroform extraction, and amplified by TLAD.
|
Label |
Cy3
|
Label protocol |
The DNA were covalently coupled separately with either Cy5 or Cy3 monoreactive fluors in 50 mM sodium bicarbonate, pH 9.0. The Cy5 and Cy3 labelled cDNAs were combined and purified with a Qiagen MinElute ERC cleanup kit and suspended in 50 µl of 3.4X SSC, 2 mg/ml of yeast tRNA, and 0.3% SDS for hybridization to the microarray.
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|
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Channel 2 |
Source Name |
40 min
|
Organism(s) |
Saccharomyces cerevisiae |
Characteristics |
Strain: BY4741 bar1(del)
|
Extracted molecule |
genomic DNA |
Extraction protocol |
DNA was isolated from mononucleosomal cell extract via proteinase K digestion and phenol:chloroform extraction, and amplified by TLAD.
|
Label |
Cy5
|
Label protocol |
The DNA were covalently coupled separately with either Cy5 or Cy3 monoreactive fluors in 50 mM sodium bicarbonate, pH 9.0. The Cy5 and Cy3 labelled cDNAs were combined and purified with a Qiagen MinElute ERC cleanup kit and suspended in 50 µl of 3.4X SSC, 2 mg/ml of yeast tRNA, and 0.3% SDS for hybridization to the microarray.
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|
|
|
Hybridization protocol |
Amplified, labeled ChIP DNA were mixed, purified by microcon, and hybridized to the microarrays in 3.4X SSC and 0.3% SDS for 16 hours at 55 degrees.
|
Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000B fluorescent scanner at 5 micron resolution and image intensity data were extracted and analyzed with GenePix Pro 5.1 analysis software.
|
Description |
BY4741 cells bearing a deletion in bar1 were grown to an OD of 0.4 and arrested by adding alpha factor to a final concentration of 1 ug/ml and incubated for 3 hours. To release from arrest, cells were filtered with 0.22 uM Whatman filter, washed with sterile MilliQ water, and the filter paper placed in a flask prewarmed to 30C, containing 450 ml YPD and 22.5 mg pronase. Upon release, cells were harvested after 40 minutes, crosslinked with formaldehyde (1%), washed with water and zymolyase treated. Extracts of these cells were then digested with MNase (ChIP Input). As a reference, DNA was obtained from sonication of unsynchronized cells. The ChIP and reference samples were amplified via TLAD and labeled with cy5 and cy3 respectively, and co-hybridized to our custom nucleosomal arrays which contain oligos spanning chromosome III as well as select promoter regions from other chromosomes.
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Data processing |
After background correction and removal of flagged values, data was block normalized by dilation and log base 2 ratios (Cy5/Cy3) were calculated to produce the values given in the data table.
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Submission date |
Jan 23, 2009 |
Contact name |
Oliver Rando |
E-mail(s) |
Oliver.Rando@umassmed.edu
|
Phone |
508-856-8879
|
Fax |
1-508-856-6464
|
Organization name |
UMass Medical School
|
Street address |
364 Plantation St.
|
City |
Worcester |
State/province |
MA |
ZIP/Postal code |
01605-4321 |
Country |
USA |
|
|
Platform ID |
GPL2625 |
Series (1) |
GSE14565 |
Lack of epigenetic maintenance of active chromatin states in budding yeast |
|
Data table header descriptions |
ID_REF |
Probe ID |
Block |
printing block |
VALUE |
Block Normalized Log2 Ratio (635/532) |
Raw Log Ratio (635/532) |
Raw log (base 2) transform of the ratio (635/532) of the medians, as calculated by GenePix |
Diameter |
diameter in microns of the array feature |
F635 Median |
median feature pixel intensity at wavelength 635 nm |
F635 Mean |
mean feature pixel intensity at wavelength 635 nm |
F635 SD |
standard deviation of the feature pixel intensity at wavelength 635 nm |
F635 CV |
the coefficient of variation of the feature pixel intensity at wavelength 635 nm |
B635 |
the background value that is used for any background subtraction at wavelength 635 nm, determined from local feature background median at wavelength 635 nm |
B635 Median |
median feature pixel background intensity at wavelength 635 nm |
B635 Mean |
mean feature pixel background intensity at wavelength 635 nm |
B635 SD |
standard deviation of the background feature pixel intensity at wavelength 635 nm |
B635 CV |
the coefficient of variation of the local background intensity at wavelength 635 nm |
% > B635+1SD |
the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 635 nm |
% > B635+2SD |
the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 635 nm |
F635 % Sat. |
the percentage of feature pixels at wavelength 635 nm that are saturated |
F532 Median |
median feature pixel intensity at wavelength 532 nm |
F532 Mean |
mean feature pixel intensity at wavelength 532 nm |
F532 SD |
standard deviation of the feature pixel intensity at wavelength 532 nm |
F532 CV |
the coefficient of variation of the feature pixel intensity at wavelength 532 nm |
B532 |
the background value that is used for any background subtraction at wavelength 532 nm, determined from local feature background median at wavelength 532 nm |
B532 Median |
median feature pixel background intensity at wavelength 532 nm |
B532 Mean |
mean feature pixel background intensity at wavelength 532 nm |
B532 SD |
standard deviation of the background feature pixel intensity at wavelength 532 nm |
B532 CV |
the coefficient of variation of the local background intensity at wavelength 532 nm |
% > B532+1SD |
the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 532 nm |
% > B532+2SD |
the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm |
F532 % Sat. |
the percentage of feature pixels at wavelength 532 nm that are saturated |
Ratio of Medians (635/532) |
the ratio (635/532) of the median intensities of each feature for each wavelength, with the median background subtracted |
Ratio of Means (635/532) |
the ratio (635/532) of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted |
Median of Ratios (635/532) |
the median of pixel-by-pixel ratios (635/532) of pixel intensities, with the median background subtracted |
Mean of Ratios (635/532) |
the geometric mean of the pixel-by-pixel ratios (635/532) of pixel intensities, with the median background subtracted |
Ratios SD (635/532) |
the geometric standard deviation of the pixel intensity ratios |
Rgn Ratio (635/532) |
the regression ratio (635/532) of every pixel in a 2-feature-diameter circle around the center of the feature |
Rgn R2 (635/532) |
the coefficient of determination for the current regression value |
F Pixels |
the total number of feature pixels |
B Pixels |
the total number of background pixels |
Circularity |
a metric based on the variance of the distance of each boundary pixel to the centroid of the feature: 100 is most circular, 0 is most non-circular |
Sum of Medians (635/532) |
the sum of the median intensities for each wavelength, with the median background subtracted |
Sum of Means (635/532) |
the sum of the arithmetic mean intensities for each wavelength, with the median background subtracted |
F635 Median - B635 |
the median feature pixel intensity at wavelength 635 nm with the median background subtracted |
F532 Median - B532 |
the median feature pixel intensity at wavelength 532 nm with the median background subtracted |
F635 Mean - B635 |
the mean feature pixel intensity at wavelength 635 nm with the median background subtracted |
F532 Mean - B532 |
the mean feature pixel intensity at wavelength 532 nm with the median background subtracted |
F635 Total Intensity |
the sum of feature pixel intensities at wavelength 635 nm |
F532 Total Intensity |
the sum of feature pixel intensities at wavelength 532 nm |
SNR 635 |
the signal-to-noise ratio at wavelength 635 nm, defined by (Mean Foreground 635- Mean Background 635) / (Standard deviation of Background 635) |
SNR 532 |
the signal-to-noise ratio at wavelength 532 nm, defined by (Mean Foreground 532- Mean Background 532) / (Standard deviation of Background 532) |
Flags |
the type of flag associated with a feature; 100 = good, bad = -100, not found = -50, absent = -75, unflagged = 0 |
Autoflag |
flag used to mark whether or not a good or bad flag was applied via a number of criteria defined in boolean terms; 1 = flagged, 0 = not flagged |