CB15N cells were grown in M2GX (0.2% glucose, 0.3% xylose) to OD660 ~= 0.3 and subjected to a mock synchrony protocol that did not separate out the cell cycle stages. Specificially, cells were pelleted by centrifuging for 10 minutes at 7,740 g. The cells were then washed three times with 4°C 1X M2. Each wash was followed by a 10 minute spin at 7,740 g. Cells were then suspended in fresh 30°C M2G. After 70 minutes, a sample was taken. After 74 minutes, xylose was added to 0.3%. Then, 105 minutes after the cells were placed into fresh M2G, a second sample was taken. This channel came from the 105 min sample.
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.
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. Qiagen's PCR clean-up kit was used according to the manufacturer's directions except that 10, rather than 5, sample volumes of PB were added to ensure that the pH was correct for efficient DNA binding to the membrane. 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.
CB15N cells were grown in M2GX (0.2% glucose, 0.3% xylose) to OD660 ~= 0.3 and subjected to a mock synchrony protocol that did not separate out the cell cycle stages. Specificially, cells were pelleted by centrifuging for 10 minutes at 7,740 g. The cells were then washed three times with 4°C 1X M2. Each wash was followed by a 10 minute spin at 7,740 g. Cells were then suspended in fresh 30°C M2G. After 70 minutes, a sample was taken. After 74 minutes, xylose was added to 0.3%. Then, 105 minutes after the cells were placed into fresh M2G, a second sample was taken. This channel came from the 105 min sample.
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.
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. Qiagen's PCR clean-up kit was used according to the manufacturer's directions except that 10, rather than 5, sample volumes of PB were added to ensure that the pH was correct for efficient DNA binding to the membrane. 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 compares expression in M2G and M2GX.
Data processing
Data was filtered and normalized as described in PMID 14973021.
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
