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Sample GSM71236 Query DataSets for GSM71236
Status Public on Dec 08, 2005
Title pxylX_dnaA_cell_cycle_#2_60min_rep1
Sample type RNA
 
Channel 1
Source name CB15N
Organism Caulobacter vibrioides
Characteristics Asynchronous log phase culture of CB15N cells grown in M2GX at 30°C to OD660 = 0.34.
Extracted molecule total RNA
Extraction protocol For RNA extraction, ~6 ml of cell culture was pelleted by spinning for 1 minute in a microfuge at 14,500 g. (Each 6 ml sample was split among three 2 ml tubes.) After removing the supernatant, the pellets were frozen in liquid nitrogen. Later, 350 µl of Trizol (Invitrogen) was added to each pellet. The three parts were combined to make a single sample with a volume of ~1 ml and were incubated for 10 minutes at 65ºC to lyse the cells. Next, 200 µl of chloroform was added and samples were mixed vigorously and incubated at room temperature for 5 minutes. Phases were then separated by centrifugation (15 minutes, 4ºC, 14,500 g) and the aqueous phase, containing the RNA, was transferred to a new tube. RNA was precipitated with 0.5 ml of isopropanol at -80ºC or -20°C overnight, pelleted (30 minutes, 4ºC, 14,500 g), washed with -20°C 70% ethanol, and resuspended in 50 µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2 µl DNase I (Ambion, 2 U/µl) and 5 µl 10X DNase I buffer and incubated at 37°C for two hours. RNA was isolated using an acid phenol extraction. Specifically, 150 µl DEPC-treated water and 200 µl acid phenol chloroform (5:1, pH 4.5, Ambion) were added and the mixture was vortexed vigorously. Then, samples were spun at 14,500 g at 4°C for 10 minutes to separate the phases. The aqueous (upper) phase was transfered to a new tube. RNA was precipitated by adding 20 µl 3 M sodium acetate (pH 5.2) and 500 µl -20°C ethanol and freezing overnight at -80C. RNA was then pelleted by centrifuging at 4°C at 14,500 g for 10 minutes. Pellets were washed in 1 ml 70% ethanol, spun for 5 minutes at 4°C at 14,500 g. Supernatant was poured out, samples were spun for 30 seconds at 14,500 g at room temperature, the residual ethanol was removed with a pipet. Samples were air dried for ~10 minutes and then were suspended in 20 µl Nuclease-free water (Ambion). To aid in dissolving the RNA, samples were put at 55°C for 10 minutes or kept at room temperature for 30 minutes. RNA was stored at -80°C or -20°C. RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer. In order to obtain enough RNA to allow the same reference samples to be used for many arrays, multiple samples were taken, processed independently, and pooled.
Label Cy5
Label protocol RNA was directly labeled for microarray hybridization by reverse transcription. 20 µg RNA and 3 µg random hexamer primers (Invitrogen) in a total volume of 15 µl were heated to 65ºC for 10 minutes and cooled on ice. Next, the sample was brought to a final volume of 30 µl with the following final concentrations: 10 mM DTT, 1 x Superscript RT buffer, 0.5 mM each of ATP, GTP, TTP, 0.2 mM CTP, 400 U Superscript II reverse transcriptase (Invitrogen) and 0.1 mM of Cy5-dCTP (Amersham). The reaction was incubated at room temperature for 10 minutes and then at 42ºC for 1 hour and 50 minutes. After reverse transcription, the RNA was degraded by addition of 1.5 µl of 1 N NaOH and incubation at 65ºC for 10 minutes; the reaction was neutralized immediately by adding 1.5 µl 1 N HCl. The cDNA from the two samples to be compared were combined and purified using Qiagen's PCR purification kit. Samples were eluted in EB buffer and were concentrated to less than 5 µl using Microcon-30 columns (Millipore). Sample volume was then brought up to 5 µl using EB. A hybridization mixture containing the labeled cDNAs, 20 µg yeast tRNA, 5X SSC, 0.1% SDS and 30% formamide in a final volume of 15 µl was incubated for 1 minute at 100ºC. After allowing the sample to cool to 42ºC for 1 minute, 12 µl of the mixture was placed on a 22x22 mm coverslip and the microarray slide was placed on the coverslip. Then, 12 µl of drops of 3X SSC were placed at the edges of the slide. The slide was quickly put into a sealed hybridization chamber, which was placed in a 44 ºC water bath overnight. Prior to use, slides were blocked by shaking sequentially in 0.2% SDS (2 minutes, 2 times), distilled water (2 minutes, 2 times), aldehyde blocking solution (0.75 g sodium borohydride, 225 ml 1X PBS, 75 ml 95% ethanol, 15 minutes), 0.2% SDS (2 minutes, 2 times), and water (2 minutes, 2 times) and then dried by spinning at 1000 rpm for 2 minutes in a Sorvall Legend RT tabletop centrifuge. Following hybridization, arrays were washed for 10 minutes at 37ºC in 2X SSC, 0.2% SDS, followed by a 10 minute wash in 2X SCC and a 10 minute wash in 0.2X SSC, both at room temperature. Slides were then dried by spinning at 1000 rpm in a Sorvall Legend RT tabletop centrifuge for 2 minutes at room temperature.
 
Channel 2
Source name LS3350 (GM2471) del dnaA, PxylX::dnaA
Organism Caulobacter vibrioides
Characteristics Swarmer-stage cells were isolated from a 30°C log phase M2GX (0.2% glucose, 0.3% xylose) culture and released into M2G at 30°C. After 74 minutes, dnaA expression was induced by adding xylose to 0.3%. Samples were taken every 15 minutes. This sample was taken at 60 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73
Extracted molecule total RNA
Extraction protocol For RNA extraction, ~6 ml of cell culture was pelleted by spinning for 1 minute in a microfuge at 14,500 g. (Each 6 ml sample was split among three 2 ml tubes.) After removing the supernatant, the pellets were frozen in liquid nitrogen. Later, 350 µl of Trizol (Invitrogen) was added to each pellet. The three parts were combined to make a single sample with a volume of ~1 ml and were incubated for 10 minutes at 65ºC to lyse the cells. Next, 200 µl of chloroform was added and samples were mixed vigorously and incubated at room temperature for 5 minutes. Phases were then separated by centrifugation (15 minutes, 4ºC, 14,500 g) and the aqueous phase, containing the RNA, was transferred to a new tube. RNA was precipitated with 0.5 ml of isopropanol at -80ºC or -20°C overnight, pelleted (30 minutes, 4ºC, 14,500 g), washed with -20°C 70% ethanol, and resuspended in 20 µl Nuclease-free water (Ambion). To aid in dissolving the RNA, samples were put at 55°C for 10 minutes or kept at room temperature for 30 minutes. RNA was stored at -80°C or -20°C. RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer. Since the same reference samples were used for many arrays, multiple samples were taken, processed independently, and pooled.
Label Cy3
Label protocol RNA was directly labeled for microarray hybridization by reverse transcription. 20 µg RNA and 3 µg random hexamer primers (Invitrogen) in a total volume of 15 µl were heated to 65ºC for 10 minutes and cooled on ice. Next, the sample was brought to a final volume of 30 µl with the following final concentrations: 10 mM DTT, 1 x Superscript RT buffer, 0.5 mM each of ATP, GTP, TTP, 0.2 mM CTP, 400 U Superscript II reverse transcriptase (Invitrogen) and 0.1 mM of Cy3-dCTP (Amersham). The reaction was incubated at room temperature for 10 minutes and then at 42ºC for 1 hour and 50 minutes. After reverse transcription, the RNA was degraded by addition of 1.5 µl of 1 N NaOH and incubation at 65ºC for 10 minutes; the reaction was neutralized immediately by adding 1.5 µl 1 N HCl. The cDNA from the two samples to be compared were combined and purified using Qiagen's PCR purification kit. Samples were eluted in EB buffer and were concentrated to less than 5 µl using Microcon-30 columns (Millipore). Sample volume was then brought up to 5 µl using EB. A hybridization mixture containing the labeled cDNAs, 20 µg yeast tRNA, 5X SSC, 0.1% SDS and 30% formamide in a final volume of 15 µl was incubated for 1 minute at 100ºC. After allowing the sample to cool to 42ºC for 1 minute, 12 µl of the mixture was placed on a 22x22 mm coverslip and the microarray slide was placed on the coverslip. Then, 12 µl of drops of 3X SSC were placed at the edges of the slide. The slide was quickly put into a sealed hybridization chamber, which was placed in a 44 ºC water bath overnight. Prior to use, slides were blocked by shaking sequentially in 0.2% SDS (2 minutes, 2 times), distilled water (2 minutes, 2 times), aldehyde blocking solution (0.75 g sodium borohydride, 225 ml 1X PBS, 75 ml 95% ethanol, 15 minutes), 0.2% SDS (2 minutes, 2 times), and water (2 minutes, 2 times) and then dried by spinning at 1000 rpm for 2 minutes in a Sorvall Legend RT tabletop centrifuge. Following hybridization, arrays were washed for 10 minutes at 37ºC in 2X SSC, 0.2% SDS, followed by a 10 minute wash in 2X SCC and a 10 minute wash in 0.2X SSC, both at room temperature. Slides were then dried by spinning at 1000 rpm in a Sorvall Legend RT tabletop centrifuge for 2 minutes at room temperature.
 
 
Scan protocol Images were collected for both Cy3 (CH2) and Cy5 (CH1) with a GenePix 4000B scanner. Image intensity data were extracted using GenePix Pro 4.0 software.
Description Sample taken 60 min into the second repetition of the PxylX::dnaA cell cycle
Data processing Data was filtered and normalized as described in PMID 14973021.
 
Submission date Aug 20, 2005
Last update date Dec 08, 2005
Contact name Alison Hottes
Organization name Princeton University
Department Lewis-Sigler Institute for Integrative Genomics
Street address 220 Carl Icahn Lab Washington Road
City Princeton
State/province NJ
ZIP/Postal code 08544
Country USA
 
Platform ID GPL2749
Series (1)
GSE3171 DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus

Data table header descriptions
ID_REF unique identifer for locations on platform
DIAMETER The diameter in micrometers of the spot
F635_MEDIAN CH1 (635 nm) median fluorescence intensity
F635_MEAN CH1 (635 nm) mean fluorescence intensity
F635_SD CH1 (635 nm) fluorescence intensity standard deviation
B635_MEDIAN CH1 (635 nm) background median fluorescence intensity
B635_MEAN CH1 (635 nm) background mean fluorescence intensity
B635_SD CH1 (635 nm) background fluorescence intensity standard deviation
PERCENT_GT_B635_PLUS_1SD percentage of feature pixels with a CH1 (635 nm) value greater than one standard deviations over the background in CH1 (635 nm)
PERCENT_GT_B635_PLUS_2SD percentage of feature pixels with a CH1 (635 nm) value greater than two standard deviations over the background in CH1 (635 nm)
F635_PER_SAT percentage of feature pixels at saturated level for CH1 (635 nm)
F532_MEDIAN CH2 (532 nm) median fluorescence intensity
F532_MEAN CH2 (532 nm) mean fluorescence intensity
F532_SD CH2 (532 nm) fluorescence intensity standard deviation
B532_MEDIAN CH2 (532 nm) background median fluorescence intensity
B532_MEAN CH2 (532 nm) background mean fluorescence intensity
B532_SD CH2 (532 nm) background fluorescence intensity standard deviation
PERCENT_GT_B532_PLUS_1SD percentage of feature pixels with a CH2 (532 nm) value greater than one standard deviations over the background for CH2 (532 nm)
PERCENT_GT_B532_PLUS_2SD percentage of feature pixels with a CH2 (532 nm) value greater than two standard deviations over the background
F532_PERCENT_SAT percentage of feature pixels at saturated level for CH2 (532 nm)
RATIOS_SD the standard deviation of the log of pixel intensity ratios for channels 1 and 2. Note: ratios greater than 100 and less than 0.01 are excluded when calculating this data type.
RGN_RATIO the regression ratio for channels 1 and 2 (slope of the line of best fit (chi-square or minimum-of-squares method) through the set of pixels in a circle of diameter twice that of the feature-indicator of the current feature).
RGN_R2 the coefficient of determination for the regression value for channels 1 and 2
F_PIXELS the total number of feature pixels
B_PIXELS the total number of local background pixels
FLAGS flag used to mark the quality of a feature
MED_OF_RATIO the median of pixel-by-pixel ratios of pixel intensities for channels 1 and 2, with the median background intensity subtracted
MEAN_OF_RATIO the geometric mean of the pixel-by-pixel ratios of pixel intensities for channels 1 and 2, with the median background intensity subtracted
VALUE log2(Channel1/channel2) ratio.

Data table
ID_REF DIAMETER F635_MEDIAN F635_MEAN F635_SD B635_MEDIAN B635_MEAN B635_SD PERCENT_GT_B635_PLUS_1SD PERCENT_GT_B635_PLUS_2SD F635_PER_SAT F532_MEDIAN F532_MEAN F532_SD B532_MEDIAN B532_MEAN B532_SD PERCENT_GT_B532_PLUS_1SD PERCENT_GT_B532_PLUS_2SD F532_PERCENT_SAT RATIOS_SD RGN_RATIO RGN_R2 F_PIXELS B_PIXELS FLAGS MED_OF_RATIO MEAN_OF_RATIO VALUE
145419 105 139 141 43 22 24 11 100 99 0 115 116 25 30 31 10 100 100 0 1.665 1.482 .718 316 2183 0 1.38 1.33 0.787321649278084
145423 105 181 188 57 22 23 9 100 100 0 224 230 60 30 31 8 100 100 0 1.468 .836 .814 316 2272 0 .833 .811 -0.0284855485034806
145427 105 1017 1025 158 22 23 8 100 100 0 1758 1746 323 30 31 9 100 100 0 1.223 .566 .933 316 2247 0 .59 .588 -0.70077024323577
145431 100 62 65 26 23 24 9 92 82 0 94 95 20 31 31 9 100 99 0 2.063 .711 .488 316 2255 0 .653 .607 -0.220197852679962
145435 105 713 762 239 22 24 14 100 100 0 869 883 257 31 33 18 100 100 0 1.267 .878 .918 316 2338 0 .874 .861 -0.132822604785601
145439 100 559 552 160 22 24 9 100 100 0 450 449 105 30 30 8 100 100 0 1.359 1.309 .898 316 2258 0 1.275 1.238 0.417770635931756
145515 110 267 264 79 22 24 9 100 99 0 305 298 66 30 31 9 100 99 0 1.497 .922 .87 392 2345 0 .906 .873 -0.0179824636822961
145519 105 343 349 78 22 24 15 100 100 0 275 279 58 30 31 9 100 100 0 1.347 1.34 .87 316 2286 0 1.322 1.314 0.489533614695562
145523 110 103 104 33 22 24 11 99 98 0 112 111 27 29 31 11 99 98 0 1.71 1.044 .697 392 2380 0 1 .969 0.223246466196785
145527 105 1882 1859 462 23 25 10 100 100 0 2495 2501 517 30 31 8 100 100 0 1.227 .751 .921 316 2150 0 .733 .735 -0.515199105907768
145531 110 341 345 98 23 25 10 100 100 0 251 248 63 31 31 8 100 99 0 1.378 1.5 .885 392 2287 0 1.471 1.488 0.626485531320637
145535 100 341 336 95 23 24 9 100 100 0 359 355 87 30 31 8 100 99 0 1.489 .973 .879 316 2237 0 .982 .959 -0.0437193453001179
145611 105 432 439 107 24 25 12 100 100 0 519 534 137 31 32 11 100 100 0 1.288 .814 .901 316 2254 0 .835 .828 -0.316540281493016
145615 115 833 863 383 24 25 10 100 100 0 693 709 265 31 32 11 100 100 0 1.353 1.286 .892 392 2302 0 1.194 1.199 0.227529153230553
145619 100 25 28 10 24 25 9 22 10 0 35 36 11 30 31 10 29 9 0 3.354 1.027 .011 316 2359 -50 .909 .854
145623 105 499 501 123 24 25 9 100 100 0 463 461 86 31 32 9 100 100 0 1.281 1.139 .897 316 2265 0 1.1 1.092 0.055726405861987
145627 110 472 466 122 24 25 9 99 99 0 355 353 79 31 32 8 100 100 0 1.496 1.412 .897 392 2300 0 1.382 1.333 0.356143322024844
145631 110 1564 1553 351 24 25 9 100 100 0 1569 1606 378 31 31 8 100 100 0 1.301 .962 .932 392 2312 0 .974 .966 -0.246812156609784
145707 105 409 419 106 24 25 10 100 100 0 421 417 88 30 31 8 100 100 0 1.278 1.044 .9 316 2287 0 1.004 1.01 -0.112912593609977
145711 110 1289 1302 467 24 25 9 100 100 0 1039 1068 390 31 31 8 100 99 0 1.477 1.231 .933 392 2412 0 1.238 1.263 0.0849008214857301

Total number of rows: 3842

Table truncated, full table size 474 Kbytes.




Supplementary data files not provided

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