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Sample GSM562238 Query DataSets for GSM562238
Status Public on Jul 16, 2010
Title Pol2 IP from rpb1-1 cells 120 min with diamide [time course 3]
Sample type other
 
Channel 1
Source name Pol2 IP
Organism Saccharomyces cerevisiae
Characteristics strain: BY4741 rpb1-1 (TS)
genotype: rpb1-1 mutant
stress: diamide
time: 120 min
antibody: anti-Rpb3 monoclonal antibody (Neoclone; catalog# W0012)
Extracted molecule genomic DNA
Extraction protocol Chromatin associated with the target epitope was immunoprecipitated from mononucleosomal cell extract and amplified by TLAD.
Label Cy3-dCTP
Label protocol 3 ul of amplified DNA was combined with 20 ul random octamers from the Invitrogen BioPrime Labeling Kit and nuclease-free water to a volume of 41 ul. This was boiled for 5 minutes at 100C, then incubated on ice for 5 minutes. 5 ul of a low-C dNTP mix (25 mM dATP, 15 mM dCTP, 25 mM dGTP and 25 mM dTTP) and 3 ul of 1 mM Cy3-dCTP (for IP) or Cy5-dCTP (for input) were added, along with 1 ul of exo- Klenow fragment DNA polymerase (from the BioPrime kit), and the samples were incubated for 2.5 hours at 37C. IP and input probes were combined and brought up to 500 ul volume with water in a YM-30 Microcon column, and spun at 14,000 x g until the volume reduced to <50 ul. Water was added and the spin was repeated twice, with the final volume at 37.5 ul. At this stage, samples were labeled and ready for hybridization onto Agilent 44K arrays.
 
Channel 2
Source name input
Organism Saccharomyces cerevisiae
Characteristics strain: BY4741 rpb1-1 (TS)
genotype: rpb1-1 mutant
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-dCTP
Label protocol 3 ul of amplified DNA was combined with 20 ul random octamers from the Invitrogen BioPrime Labeling Kit and nuclease-free water to a volume of 41 ul. This was boiled for 5 minutes at 100C, then incubated on ice for 5 minutes. 5 ul of a low-C dNTP mix (25 mM dATP, 15 mM dCTP, 25 mM dGTP and 25 mM dTTP) and 3 ul of 1 mM Cy3-dCTP (for IP) or Cy5-dCTP (for input) were added, along with 1 ul of exo- Klenow fragment DNA polymerase (from the BioPrime kit), and the samples were incubated for 2.5 hours at 37C. IP and input probes were combined and brought up to 500 ul volume with water in a YM-30 Microcon column, and spun at 14,000 x g until the volume reduced to <50 ul. Water was added and the spin was repeated twice, with the final volume at 37.5 ul. At this stage, samples were labeled and ready for hybridization onto Agilent 44K arrays.
 
 
Hybridization protocol Amplified ChIP and ChIP input were mixed, purified by microcon, and hybridized to the microarrays in Agilent hybridization buffer for 16 hours at 65 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 6.0 analysis software.
Description BY4741 cells bearing a temperature-sensitive mutation in rpb1were grown to an OD of 0.5 in YPD shaking at 200 rpm at room temperature (25 degrees C). The cells were treated with diamide (1.5 mM final concentration) for the indicated time before harvesting. Cells were then crosslinked with formaldehyde (1%) and washed with water. Cells were sonicated to shear DNA to ~500 bp, and subjected to immunoprecipitation with 5 ul anti-Rpb3 monoclonal antibody. As a reference, DNA was obtained from the input sheared chromatin prior to antibody addition. The ChIP and reference samples were amplified via Round A Round B amplification and labeled with cy3 and cy5 respectively, and co-hybridized to 4x44 Agilent S.cerevisiae whole genome arrays.
Data processing After background correction and removal of flagged values, data was block normalized by dilation and log base 2 expression ratios were calculated to produce the values given in the data table.
 
Submission date Jul 01, 2010
Last update date Jul 16, 2010
Contact name Oliver Rando
E-mail(s) Oliver.Rando@umassmed.edu
Phone 508-856-8879
Organization name UMass Medical School
Street address 364 Plantation St.
City Worcester
State/province MA
ZIP/Postal code 01605-4321
Country USA
 
Platform ID GPL4131
Series (1)
GSE22675 RNA Polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast

Data table header descriptions
ID_REF Probe ID
Block printing block
VALUE Block Normalized Log2 (635/532) Ratio
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

Data table
ID_REF Block VALUE Raw Log Ratio (635/532) Diameter F635 Median F635 Mean F635 SD F635 CV B635 B635 Median B635 Mean B635 SD B635 CV % > B635+1SD % > B635+2SD F635 % Sat. F532 Median F532 Mean F532 SD F532 CV B532 B532 Median B532 Mean B532 SD B532 CV % > B532+1SD % > B532+2SD F532 % Sat. Ratio of Medians (635/532) Ratio of Means (635/532) Median of Ratios (635/532) Mean of Ratios (635/532) Ratios SD (635/532) Rgn Ratio (635/532) Rgn R2 (635/532) F Pixels B Pixels Circularity Sum of Medians (635/532) Sum of Means (635/532) F635 Median - B635 F532 Median - B532 F635 Mean - B635 F532 Mean - B532 F635 Total Intensity F532 Total Intensity SNR 635 SNR 532 Flags Autoflag
23152 1 -0.49361635 -0.568 55 23853 21689 8006 36 67 67 67 12 17 100 100 0 35323 37061 14846 40 59 59 61 24 39 100 100 0 0.675 0.584 0.635 0.564 1.529 0.558 0.900 96 9 82 59050 58624 23786 35264 21622 37002 2082102 3557864 1801.833 1541.667 0 0
20038 1 -0.66361635 -0.738 55 33593 27532 12673 46 50 50 52 15 28 100 100 0 56000 48731 16899 34 56 56 60 17 28 100 100 7 0.600 0.565 0.597 0.485 1.657 0.594 0.962 94 52 82 89487 76157 33543 55944 27482 48675 2588013 4580739 1832.000 2863.000 0 0
3144 1 -0.10861635 -0.183 55 8720 7797 2592 33 56 56 59 23 38 100 100 0 9888 9308 3439 36 54 54 51 13 25 100 100 0 0.881 0.837 0.819 0.876 1.718 0.809 0.904 94 16 88 18498 16995 8664 9834 7741 9254 732884 874963 336.435 712.077 0 0
41483 1 -0.92061635 -0.995 55 7543 6664 2401 36 51 51 48 15 31 100 100 0 14992 13677 4780 34 60 60 64 11 17 100 100 0 0.502 0.486 0.494 0.491 1.817 0.471 0.908 93 6 89 22424 20230 7492 14932 6613 13617 619777 1271976 441.067 1237.545 0 0
9912 1 -0.63061635 -0.705 50 17497 15585 4387 28 37 37 41 12 29 100 100 0 28528 25868 8104 31 65 65 60 15 25 100 100 0 0.613 0.603 0.590 0.619 1.290 0.587 0.976 90 9 93 45923 41351 17460 28463 15548 25803 1402640 2328084 1295.333 1720.533 0 0
5093 1 -0.47161635 -0.546 55 4131 3753 1509 40 49 49 53 23 43 100 100 0 6003 5945 1961 32 44 44 46 12 26 100 100 0 0.685 0.628 0.666 0.568 1.674 0.647 0.933 95 13 96 10041 9605 4082 5959 3704 5901 356493 564812 160.870 491.583 0 0
17702 1 -1.04461635 -1.119 55 4153 3480 1446 41 46 46 49 13 26 100 100 0 8977 7943 2754 34 59 59 57 12 21 100 100 0 0.461 0.436 0.456 0.383 2.805 0.410 0.763 96 13 87 13025 11318 4107 8918 3434 7884 334119 762570 263.923 657.167 0 0
35319 1 -1.17961635 -1.254 50 4185 3742 1104 29 36 36 56 36 64 100 100 0 9952 8943 2802 31 54 54 56 12 21 100 100 0 0.419 0.417 0.411 0.424 1.262 0.405 0.972 87 10 93 14047 12595 4149 9898 3706 8889 325592 778051 102.389 740.583 0 0
21415 1 -1.14361635 -1.218 55 4736 4012 1900 47 49 49 53 18 33 100 98 0 10962 10324 3389 32 58 58 56 7 12 100 100 0 0.430 0.386 0.419 0.318 2.223 0.402 0.872 97 9 95 15591 14229 4687 10904 3963 10266 389167 1001466 219.944 1466.857 0 0
18721 1 -1.18861635 -1.263 50 5240 4711 1417 30 52 52 53 13 24 100 100 0 12500 11407 3277 28 53 53 59 15 25 100 100 0 0.417 0.410 0.414 0.401 1.635 0.394 0.884 89 12 84 17635 16013 5188 12447 4659 11354 419258 1015264 358.308 756.533 0 0
15134 1 -1.06161635 -1.136 55 5404 4652 2356 50 52 52 55 17 30 100 97 0 11820 10668 4213 39 60 60 61 19 31 100 100 0 0.455 0.434 0.463 0.326 3.405 0.422 0.763 101 10 85 17112 15208 5352 11760 4600 10608 469842 1077473 270.412 558.263 0 0
12371 1 -0.57561635 -0.650 50 4606 4178 1148 27 43 43 49 15 30 100 100 0 7213 6817 1752 25 51 51 52 14 26 100 100 0 0.637 0.611 0.612 0.605 1.547 0.597 0.915 89 9 92 11725 10901 4563 7162 4135 6766 371848 606671 275.267 483.214 0 0
15527 1 -0.64061635 -0.715 50 4748 4444 1082 24 55 55 51 11 21 100 100 0 7763 7016 2122 30 59 59 59 10 16 100 100 0 0.609 0.631 0.621 0.658 1.402 0.605 0.950 89 9 90 12397 11346 4693 7704 4389 6957 395557 624429 399.364 695.700 0 0
38311 1 -0.89061635 -0.965 50 4019 3732 1302 34 48 48 46 12 26 100 100 0 7798 7519 2233 29 48 48 47 11 23 100 100 0 0.512 0.493 0.517 0.465 1.585 0.492 0.908 90 21 92 11721 11155 3971 7750 3684 7471 335910 676673 307.167 679.273 0 0
36251 1 -0.76861635 -0.843 50 4939 4538 1336 29 60 60 61 19 31 100 100 0 8807 8117 2331 28 57 57 56 16 28 100 100 0 0.558 0.556 0.560 0.549 1.397 0.546 0.951 90 22 91 13629 12538 4879 8750 4478 8060 408410 730513 235.632 503.813 0 0
3944 1 -0.54361635 -0.618 55 4292 4159 1285 30 87 87 77 25 32 100 100 0 6516 6086 2735 44 61 61 60 12 20 100 100 0 0.651 0.676 0.629 0.780 2.058 0.599 0.842 93 9 79 10660 10097 4205 6455 4072 6025 386788 565977 163.280 502.167 0 0
17109 1 -0.77761635 -0.852 55 5435 4946 1461 29 56 56 56 16 28 100 100 0 9774 8916 3021 33 62 62 66 14 21 100 100 0 0.554 0.552 0.539 0.559 1.551 0.528 0.931 91 11 88 15091 13744 5379 9712 4890 8854 450107 811395 305.625 632.143 0 0
42414 1 -0.78461635 -0.859 55 3006 2682 1228 45 49 49 49 20 40 100 98 0 5420 5196 1964 37 58 58 55 15 27 100 100 0 0.551 0.512 0.550 0.438 2.871 0.483 0.713 96 12 89 8319 7771 2957 5362 2633 5138 257468 498783 131.650 342.733 0 0
27484 1 -0.99661635 -1.071 55 3846 3092 1531 49 58 58 56 18 32 95 94 0 8016 7309 2160 29 59 59 59 19 32 100 100 0 0.476 0.418 0.465 0.313 3.303 0.439 0.805 98 8 89 11745 10284 3788 7957 3034 7250 303047 716289 168.667 381.579 0 0
12031 1 -0.58261635 -0.657 50 4437 3834 1315 34 43 43 49 17 34 100 100 0 6977 6151 2099 34 48 48 48 8 16 100 100 0 0.634 0.621 0.629 0.613 1.616 0.608 0.924 94 12 94 11323 9894 4394 6929 3791 6103 360417 578237 222.647 762.875 0 0

Total number of rows: 41775

Table truncated, full table size 9637 Kbytes.




Supplementary file Size Download File type/resource
GSM562238.gpr.gz 5.7 Mb (ftp)(http) GPR
Processed data included within Sample table
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