Asynchronous log phase culture grown in M2G at 30°C to OD660 = 0.34
Extracted molecule
total RNA
Extraction protocol
For RNA extraction, 2 ml of cell culture was pelleted by spinning for 1 minute in a microfuge at 14,500 g. After removing the supernatant, cell pellets were frozen in liquid nitrogen. Later 350 µl of Trizol (Invitrogen) was added to each pellet. Three samples 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 for at least one hour, 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 -80°C. 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, and 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, at room temperature for 30 minutes, at 4°C for 4 hours. RNA was stored at -80°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
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 0.1mM of Cy5-dCTP (Amersham). The reaction was incubated at room temperature for 10 minutes and then at 42ºC for 2 hours. 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 22x22mm coverslip and the microarray slide was placed on the coverslip. 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. The slide was quickly put into a sealed hybridization chamber, which was placed in a 44ºC 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. 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.
A mixed culture of LS3313 (DivK-D90G-cold sensitive) cells was grown in M2G at 37°C to OD660 = ~0.60. Swarmer-stage cells were isolated and released into M2G at 18°C (restrictive temperature). Samples were taken hourly from 0 to 5 hours. Then, the culture was shifted to 33°C (permissive temperature). Samples were taken every 10 minutes from 310 minutes (after the initial release of the swarmers into 18°C M2G) to 380 minutes. This sample was taken at 5h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.
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, cell pellets were frozen in liquid nitrogen. Later, 350 µl of Trizol (Invitrogen) was added to each pellet. The three parts of each sample were combined into a single tube and 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 for at least one hour, pelleted (30 minutes, 4ºC, 14,500 g), washed with -20°C 70% ethanol, and resuspended in 20 µl Nuclease free water (Ambion). RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer. Genomic DNA was not visible on the gels.
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, 400U Superscript II reverse transcriptase (Invitrogen) and 0.1mM of Cy3-dCTP (Amersham). The reaction was incubated at room temperature for 10 minutes and then at 42ºC for 2 hours. 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 22x22mm coverslip and the microarray slide was placed on the coverslip. 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. The slide was quickly put into a sealed hybridization chamber, which was placed in a 44ºC 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. 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 5h into the DivK-cs modified cell cycle
Data processing
Data was filtered and normalized as described in PMID 14973021.
Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease
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
