NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM562240 Query DataSets for GSM562240
Status Public on Jul 16, 2010
Title Pol2 IP from rpb1-1 cells 10 min 37C heat shock, then 30 min diamide [time course 4]
Sample type other
 
Channel 1
Source name Pol2 IP
Organism Saccharomyces cerevisiae
Characteristics strain: BY4741 rpb1-1 (TS)
genotype: rpb1-1 mutant
stress: 37C heat shock, then diamide
time: 10 min heat shock, 30 min diamide
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). An equal volume of YPD prewarmed to 49 degrees C was added to flasks, which were immediately transferred to a 37 degrees C incubator and incubated at the indicated time. 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 -0.305839045 -0.220 55 23249 19517 7164 36 67 67 65 21 32 100 100 0 27058 24380 9947 40 56 56 58 17 29 100 100 0 0.859 0.800 0.786 0.833 2.045 0.766 0.885 96 10 83 50184 43774 23182 27002 19450 24324 1873609 2340504 926.286 1430.706 0 0
20038 1 -0.613839045 -0.528 55 28648 23789 9210 38 44 44 46 15 32 100 100 0 41316 35656 14502 40 62 62 61 15 24 100 100 0 0.693 0.667 0.661 0.680 1.747 0.648 0.936 94 20 96 69858 59339 28604 41254 23745 35594 2236169 3351674 1582.867 2373.000 0 0
3144 1 -0.296839045 -0.211 55 7035 6340 2054 32 60 60 62 24 38 100 100 0 8133 7657 3161 41 61 61 60 17 28 100 100 0 0.864 0.827 0.793 0.930 2.673 0.762 0.824 92 11 95 15047 13876 6975 8072 6280 7596 583290 704430 261.583 446.882 0 0
41483 1 -0.456839045 -0.371 55 5451 4834 1798 37 47 47 47 11 23 100 100 0 7040 6433 1974 30 53 53 53 13 24 100 100 0 0.773 0.750 0.759 0.705 1.694 0.763 0.910 91 12 93 12391 11167 5404 6987 4787 6380 439881 585415 435.182 490.769 0 0
9912 1 -0.197839045 -0.112 55 16754 13070 6359 48 50 50 54 22 40 100 100 0 18105 16698 4546 27 58 58 67 24 35 100 100 0 0.926 0.782 0.880 0.595 2.570 0.876 0.819 98 6 88 34751 29660 16704 18047 13020 16640 1280896 1636422 591.636 692.958 0 0
5093 1 -0.649839045 -0.564 55 3174 2795 1184 42 52 52 57 19 33 100 98 0 4683 4580 1238 27 69 69 66 14 21 100 100 0 0.677 0.608 0.664 0.496 2.182 0.638 0.852 93 12 91 7736 7254 3122 4614 2743 4511 259960 425936 144.105 322.429 0 0
17702 1 -0.300839045 -0.215 50 3120 2899 699 24 39 39 44 13 29 100 100 0 3632 3372 1113 33 57 57 54 7 12 100 98 0 0.862 0.863 0.829 0.989 2.330 0.806 0.878 90 7 93 6656 6175 3081 3575 2860 3315 260870 303473 219.615 474.000 0 0
35319 1 -0.156839045 -0.071 55 3810 3123 1480 47 52 52 57 17 29 97 96 0 4009 3789 1160 30 62 62 60 16 26 100 100 0 0.952 0.824 0.932 0.591 3.574 0.889 0.726 95 13 85 7705 6798 3758 3947 3071 3727 296724 359937 180.353 233.063 0 0
21415 1 -0.153839045 -0.068 55 5809 5317 1740 32 46 46 50 12 24 100 100 0 6095 5437 2118 38 53 53 56 9 16 100 100 0 0.954 0.979 0.959 1.123 2.643 0.937 0.847 96 9 86 11805 10655 5763 6042 5271 5384 510386 521995 438.917 597.889 0 0
18721 1 -0.404839045 -0.319 55 4396 4023 1155 28 65 65 55 20 36 100 100 0 5454 4945 1840 37 51 51 55 16 29 100 100 0 0.802 0.809 0.791 0.847 1.589 0.765 0.923 92 13 91 9734 8852 4331 5403 3958 4894 370132 454920 198.400 305.625 0 0
15134 1 -0.111839045 -0.026 55 5565 5127 1709 33 51 51 50 16 32 100 100 0 5657 5108 2128 41 43 43 46 9 19 100 100 0 0.982 1.002 0.944 1.114 2.357 0.949 0.812 97 10 86 11128 10141 5514 5614 5076 5065 497294 495428 317.313 562.444 0 0
12371 1 0.304160955 0.390 55 4234 3827 1022 26 44 44 47 11 23 100 100 0 3262 2945 1130 38 64 64 62 17 27 100 100 0 1.310 1.313 1.244 1.478 1.811 1.230 0.914 95 10 95 7388 6664 4190 3198 3783 2881 363550 279760 343.636 169.588 0 0
15527 1 0.153160955 0.239 50 4393 4132 926 22 44 44 48 16 33 100 100 0 3733 3499 1106 31 48 48 52 16 30 100 100 0 1.180 1.185 1.182 1.244 1.292 1.136 0.955 86 14 89 8034 7539 4349 3685 4088 3451 355360 300919 255.250 215.438 0 0
38311 1 -0.043839045 0.042 55 4563 4063 1369 33 46 46 47 15 31 100 100 0 4442 4016 1667 41 54 54 54 14 25 97 97 0 1.029 1.014 1.003 1.175 2.333 0.964 0.898 93 15 88 8905 7979 4517 4388 4017 3962 377879 373453 267.733 283.000 0 0
36251 1 0.034160955 0.120 50 4430 3808 1484 38 54 54 51 17 33 100 100 0 4088 3842 968 25 62 62 60 15 25 100 100 0 1.087 0.993 1.039 0.877 1.914 1.079 0.833 92 13 94 8402 7534 4376 4026 3754 3780 350329 353484 221.000 252.133 0 0
3944 1 0.052160955 0.138 50 4436 4161 1082 26 53 53 60 25 41 100 100 0 4037 3881 1039 26 54 54 51 15 29 100 100 0 1.100 1.073 1.094 1.079 1.281 1.068 0.938 90 8 95 8366 7935 4383 3983 4108 3827 374528 349316 164.040 255.333 0 0
17109 1 -0.216839045 -0.131 55 3739 3529 884 25 53 53 53 15 28 100 100 0 4101 3699 1411 38 65 65 65 18 27 100 100 0 0.913 0.957 0.915 1.063 1.768 0.880 0.887 92 14 79 7722 7110 3686 4036 3476 3634 324660 340298 231.733 201.889 0 0
42414 1 -0.140839045 -0.055 55 3149 2898 1074 37 48 48 53 22 41 100 100 0 3283 3064 1337 43 62 62 56 11 19 97 97 0 0.963 0.949 0.961 1.018 2.643 0.888 0.767 97 8 91 6322 5852 3101 3221 2850 3002 281140 297222 129.318 273.455 0 0
27484 1 -0.096839045 -0.011 55 3356 3023 932 30 46 46 47 15 31 100 100 0 3385 2993 1170 39 49 49 54 16 29 100 98 0 0.992 1.011 0.985 1.154 2.506 0.960 0.802 95 13 82 6646 5921 3310 3336 2977 2944 287190 284312 198.400 183.688 0 0
12031 1 -0.074839045 0.011 55 3345 3081 775 25 44 44 46 15 32 100 100 0 3330 2997 1071 35 55 55 52 13 25 100 100 0 1.008 1.032 1.002 1.162 1.829 0.967 0.926 91 8 92 6576 5979 3301 3275 3037 2942 280366 272737 202.333 226.538 0 0

Total number of rows: 41775

Table truncated, full table size 9476 Kbytes.




Supplementary file Size Download File type/resource
GSM562240.gpr.gz 5.6 Mb (ftp)(http) GPR
Processed data included within Sample table
| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap