title: DivK-cs cell cycle-2h-rep1

description: Sample taken 2h into the DivK-cs modified cell cycle

tag: 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 2h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-360m-rep1

status: Public on Dec 08 2005

type: RNA

city: Princeton

first: Alison

last: Hottes

state: NJ

address: 220 Carl Icahn Lab Washington Road

department: Lewis-Sigler Institute for Integrative Genomics

country: USA

institute: Princeton University

description: Sample taken 360min into the DivK-cs modified cell cycle

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.

data_processing: Data was filtered and normalized as described in PMID 14973021.

molecule: total RNA

organism: Caulobacter vibrioides

source_name: CB15N mixed culture reference

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.

extract_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.

tag: Asynchronous log phase culture grown in M2G at 30°C to OD660 = 0.34

source_name: LS3313 (divK-D90G cold-sensitive)

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.

extract_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.

tag: 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 360min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-1h-rep1

description: Sample taken 1h into the DivK-cs modified cell cycle

tag: 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 1h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: WTR40205B

city: Berkely

first: Sanchita

last: Bhattacharya

state: CA

address: 1 Cyclotron Rd

institute: MIT

description:
WildType irradiated
Replicate 2

organism: Mus musculus

source_name: Early passage primary MEFs


title: DivK-cs cell cycle-1h-rep2


title: DivK-cs cell cycle-320m-rep2

description: Sample taken 320min into the DivK-cs modified cell cycle

tag: 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 320min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: MUR40325B

description:
Mutant irradiated
Replicate 4


title: DivK-cs cell cycle-0h-rep2

description: Sample taken 0h into the DivK-cs modified cell cycle

tag: 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 0h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: Caulobacter_pye_cell_cycle_0min_rep1

description: Sample taken 0 min into a cell cycle in PYE media

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,  200 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.   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.

extract_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.  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.

tag: Asynchronous log phase culture of CB15N cells grown in PYE at 30°C to OD660 = 0.27.

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,  200 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.   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.

extract_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.

tag: Swarmer-stage cells were isolated from a 30°C log phase PYE culture and released into PYE at 30°C.  Samples were taken every 10 minutes.  This sample was taken at 0 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: MUR40323B

description:
Mutant irradiated
Replicate 3


title: MU40324B

description:
Mutant
Replicate 4


title: MU40322B

description:
Mutant
Replicate 3


title: MUR40203B

description:
Mutant irradiated
Replicate 1


title: MU40202B

description:
Mutant
Replicate1


title: WTR40321B

description:
Wild Type irradiated
Replicate 4


title: MUR40207B

description:
Mutant irradiated
Replicate2


title: WTR40201B

description:
Wild Type irradiated
Replicate1


title: WTR40319B

description:
Wild Type irradiated
Replicate 3


title: MU40206B

description:
Mutant
Replicate 2


title: DivK-cs cell cycle-0h-rep1


title: WT40200B

description:
Wild Type
Replicate 1


title: WT40320B

description:
Wild Type
Replicate 4


title: caulobacter_m2g_vs_m2gx_rep3

description: Sample compares expression in M2G and M2GX.

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.

extract_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.

tag: 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 70 min sample.

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.


title: WT40204B

description:
Wild Type
Replicate2

source_name: Early Passage of MEFs


title: WT40318B

description:
Wild Type
Replicate3


title: caulobacter_m2g_vs_m2gx_rep2

tag: 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 70 min sample.


title: caulobacter_m2g_vs_m2gx_rep4

tag: 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.


title: caulobacter_m2g_vs_m2gx_rep1

tag: 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.


title: pxylX_dnaA_cell_cycle_#2_180min_rep1

description: Sample taken 180 min into the second repetition of the PxylX::dnaA cell cycle

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.

extract_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.

tag: Asynchronous log phase culture of CB15N cells grown in M2GX at 30°C to OD660 = 0.34.

source_name: LS3350 (GM2471)  del dnaA, PxylX::dnaA

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.

tag: 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 180 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_30min_rep2

description: Sample taken 30 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 30 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_75min_rep1

description: Sample taken 75 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 75 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_30min_rep1


title: pxylX_dnaA_cell_cycle_#2_180min_rep2


title: pxylX_dnaA_cell_cycle_#2_195min_rep1

description: Sample taken 195 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 195 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_195min_rep2


title: pxylX_dnaA_cell_cycle_#2_45min_rep1

description: Sample taken 45 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 45 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_60min_rep1

description: Sample taken 60 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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


title: pxylX_dnaA_cell_cycle_#2_165min_rep1

description: Sample taken 165 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 165 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_90min_rep1

description: Sample taken 90 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 90 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_15min_rep1

description: Sample taken 15 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 15 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_45min_rep2


title: pxylX_dnaA_cell_cycle_#2_120min_rep1

description: Sample taken 120 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 120 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_150min_rep2

description: Sample taken 150 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 150 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_165min_rep2


title: pxylX_dnaA_cell_cycle_#2_150min_rep1


title: pxylX_dnaA_cell_cycle_#2_135min_rep1

description: Sample taken 135 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 135 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_120min_rep2


title: pxylX_dnaA_cell_cycle_#2_135min_rep2


title: pxylX_dnaA_cell_cycle_#2_105min_rep1

description: Sample taken 105 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 105 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pxylX_dnaA_cell_cycle_#2_0min_rep1

description: Sample taken 0 min into the second repetition of the PxylX::dnaA cell cycle

tag: 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 0 min. At 0 minutes, the OD660 was 0.29; at 195 minutes, the OD660 was 0.73


title: pleC(H610A) -rep5

description: Sample compares mRNA expression between LS3032 (CB15 wild-type) and the same strain with an allele of the pleC histidine kinase where H610 has been converted to an alanine.

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 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 5µl 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 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.

extract_protocol: For RNA extraction, ~7.5 ml of cell culture was pelleted by centrifugation for 1 minute in a microfuge at 14,500 g. (Each 7.5 ml sample was split among five 2 ml tubes.) After removing the supernatant, 150 µl of Trizol (Invitrogen) was added to each pellet.  The five 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 50 µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2 µl DNase I (Ambion, 2 U/ul) 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 centrifugation (10 minutes, 4°C,  14,500 g).   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 kept at room temperature for 30 minutes or for 4 hours at 4°C.  RNA was stored at -80°C.  RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer.

tag: Asynchronous exponential phase culture of LS3938 (CB15 pleC-H610A) grown in PYE at 30°C to OD600 = 0.28.

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 15µl 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 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 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 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.

extract_protocol: For RNA extraction, ~7.5 ml of cell culture was pelleted by centrifugation for 1 minute in a microfuge at 14,500 g. (Each 7.5 ml sample was split among five 2 ml tubes.) After removing the supernatant, 150 µl of Trizol (Invitrogen) was added to each pellet.  The five 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 50µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2 µl DNase I (Ambion, 2 U/ul) 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 20µl 3 M sodium acetate (pH 5.2) and 500µl -20°C ethanol and freezing overnight at -80°C.  RNA was then pelleted by centrifugation (10 minutes, 4°C,  14,500 g).   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 20µl Nuclease-free water (Ambion). To aid in dissolving the RNA, samples were kept at room temperature for 30 minutes or for 4 hours at 4°C.  RNA was stored at -80°C.  RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer.

tag: Asynchronous exponential phase culture of LS3032 (CB15) grown in PYE at 30°C to OD600 = 0.25.


title: pxylX_dnaA_cell_cycle_#1_90min_rep2

description: Sample taken 90 min into the first repetition of the PxylX::dnaA cell cycle

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,  300 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.

tag: Asynchronous log phase culture of CB15N cells grown in M2GX at 30°C to OD660 = 0.3727.

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,  300 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.

tag: 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 90 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pleC(H610A) -rep1

tag: Asynchronous exponential phase culture of LS3032 (CB15) grown in PYE at 30°C to OD600 = 0.27.

tag: Asynchronous exponential phase culture of LS3938 (CB15 pleC-H610A) grown in PYE at 30°C to OD600 = 0.29.


title: pxylX_dnaA_cell_cycle_#1_120min_rep1

description: Sample taken 120 min into the first repetition of the PxylX::dnaA cell cycle

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,  300 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.   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.

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,  300 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.   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.

tag: 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 120 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: divJ deletion -rep3

description: Sample compares mRNA expression between LS3032 (CB15 wild-type) and the same strain with a deletion of the divJ histidine kinase.

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 0.1 mM 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 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 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.

tag: Asynchronous exponential phase culture of LS3032 (CB15) grown in PYE at 30°C to OD600 = 0.2498.

source_name: LS4107 (CB15 divJ deletion)

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 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 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 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.

tag: Asynchronous exponential phase culture of LS4107 (CB15 divJ deletion) grown in PYE at 30°C to OD600 = 0.2505.


title: pleC(H610A) -rep3

tag: Asynchronous exponential phase culture of LS3032 (CB15) grown in PYE at 30°C to OD600 = 0.28.

tag: Asynchronous exponential phase culture of LS3939 (CB15 pleC-H610A) grown in PYE at 30°C to OD600 = 0.26.


title: pxylX_dnaA_cell_cycle_#1_60min_rep1

description: Sample taken 60 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 0.42; at 210 minutes, the OD660 was 1.1


title: divJ deletion -rep4

description: Sample compares mRNA expression between LS3032 (CB15 wild-type) and the same strain with a disruption of the divJ histidine kinase.

tag: Asynchronous exponential phase culture of LS3032 (CB15) grown in PYE at 30°C to OD600 = 0.24.

source_name: LS4117 (CB15 divJ disruption)

tag: Asynchronous exponential phase culture of LS4117 (CB15 divJ disruption) grown in PYE at 30°C to OD600 = 0.2435.


title: pxylX_dnaA_cell_cycle_#1_60min_rep2


title: pxylX_dnaA_cell_cycle_#1_90min_rep1


title: pxylX_dnaA_cell_cycle_#1_75min_rep1

description: Sample taken 75 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 75 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pleC(H610A) -rep4

tag: Asynchronous exponential phase culture of LS3939 (CB15 pleC-H610A) grown in PYE at 30°C to OD600 = 0.27.


title: pleC(H610A) -rep2


title: pxylX_dnaA_cell_cycle_#1_30min_rep1

description: Sample taken 30 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 30 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: divJ deletion -rep2

source_name: LS4110 (CB15 divJ disruption)

tag: Asynchronous exponential phase culture of LS4110 (CB15 divJ disruption) grown in PYE at 30°C to OD600 = 0.2432.


title: pxylX_dnaA_cell_cycle_#1_75min_rep2


title: pxylX_dnaA_cell_cycle_#1_210min_rep1

description: Sample taken 210 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 210 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_45min_rep1

description: Sample taken 45 min into the first repetition of the PxylX::dnaA cell cycle

tag: Asynchronous log phase culture of CB15N cells grown in M2GX at 30°C to OD660 = 0.3727.

tag: 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 45 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_0min_rep1

description: Sample taken 0 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 0 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: divJ deletion -rep1

source_name: LS4108 (CB15 divJ deletion)

tag: Asynchronous exponential phase culture of LS4108 (CB15 divJ deletion) grown in PYE at 30°C to OD600 = 0.2613.


title: pxylX_dnaA_cell_cycle_#1_150min_rep1

description: Sample taken 150 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 150 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_165min_rep1

description: Sample taken 165 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 165 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_15min_rep1

description: Sample taken 15 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 15 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_105min_rep1

description: Sample taken 105 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 105 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_105min_rep2


title: pxylX_dnaA_cell_cycle_#1_135min_rep2

description: Sample taken 135 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 135 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_120min_rep2

tag: 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 120 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: pxylX_dnaA_cell_cycle_#1_135min_rep1


title: podJ_deletion-rep4

description: Sample compares mRNA expression between LS3032 (CB15 wild-type) and the same strain with a deletion of the podJ polar organelle developmental protein.

source_name: LS3766 (CB15 del podJ)

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 2 hours. After reverse transcription, the RNA was degraded by addition of 1.5 µl of 1N 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.  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 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.

extract_protocol: For RNA extraction, ~7.5 ml of cell culture was pelleted by centrifugation for 1 minute in a microfuge at 14,500 g. (Each 7.5 ml sample was split among five 2 ml tubes.) After removing the supernatant, 150 µl of Trizol (Invitrogen) was added to each pellet.  The five 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 50 µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2ul DNase I (Ambion, 2 U/ul) 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 centrifugation (10 minutes, 4°C,  14,500 g).   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 kept at room temperature for 30 minutes or for 4 hours at 4°C.  RNA was stored at -80°C.  RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer.

tag: Asynchronous exponential phase culture of LS3766 (CB15 del podJ) grown in PYE at 30°C to OD600 = 0.2608.

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 0.1 mM 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 15µl 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 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.

extract_protocol: For RNA extraction, ~7.5 ml of cell culture was pelleted by centrifugation for 1 minute in a microfuge at 14,500 g. (Each 7.5 ml sample was split among five 2 ml tubes.) After removing the supernatant, 150 µl of Trizol (Invitrogen) was added to each pellet.  The five 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 50 µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2ul DNase I (Ambion, 2U/ul) 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 centrifugation (10 minutes, 4°C,  14,500 g).   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 kept at room temperature for 30 minutes or for 4 hours at 4°C.  RNA was stored at -80°C.  RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer.

tag: Asynchronous exponential phase culture of CB15 (LS3032) grown in PYE at 30°C to OD600 = 0.2607.


title: pxylX_dnaA_cell_cycle_#1_180min_rep1

description: Sample taken 180 min into the first repetition of the PxylX::dnaA cell cycle

tag: 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 180 min. At 0 minutes, the OD660 was 0.42; at 210 minutes, the OD660 was 1.1


title: Tcf1 knock-out pancreatic islets_replicate 1 (MG-U74B)

city: New York

first: Markus

last: Stoffel

state: NY

address: 1230 York Avenue

department: Metabolic Diseases

institute: Rockefeller University

description: Pancreatic islets total RNA

data_processing: MAS 4.0

label: Affymetrix standard protocol

source_name: Tcf1 knock out pancreatic islets

tag: Isolated pancreatic islets by collagenase digestion and ficoll gradient centrifugation


title: Tcf1 knock-out pancreatic islets_replicate 2 (MG-U74A)

description: Pancreatic islets total RNA

source_name: Tcf1 knock-out pancreatic islets


title: Wild type pancreatic islets_replicate 1 (MG-U74A)

description: Pancreatic islets total RNA

source_name: pancreatic islets


title: podJ_deletion-rep3

tag: Asynchronous exponential phase culture of LS3766 (CB15 del podJ) grown in PYE at 30°C to OD600 = 0.3963.

tag: Asynchronous exponential phase culture of CB15 (LS3032) grown in PYE at 30°C to OD600 = 0.3221.


title: podJ_deletion-rep2

tag: Asynchronous exponential phase culture of CB15 (LS3032) grown in PYE at 30°C to OD600 = 0.2446.

tag: Asynchronous exponential phase culture of LS3766 (CB15 del podJ) grown in PYE at 30°C to OD600 = 0.2628.


title: Wild type pancreatic islets_replicate 2 (MG-U74A)

description: Pancreatic islets total RNA


title: Tcf1 knock-out pancreatic islets_replicate 2 (MG-U74B)

description: Pancreatic islets total RNA


title: Wild type pancreatic islets_replicate 1 (MG-U74B)

description: Pancreatic islets total RNA

source_name: wild type pancreatic islets


title: pleC_deletion-rep2

description: Sample compares mRNA expression between LS3032 (CB15 wild-type) and the same strain with a deletion of the pleC histidine kinase.

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 was 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 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 1.5µl of 1N NaOH and incubation at 65ºC for 10 minutes; the reaction was neutralized immediately by adding 1.5 µl 1N 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 15µl 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.  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 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.

extract_protocol: For RNA extraction, ~7.5 ml of cell culture was pelleted by centrifugation for 1 minute in a microfuge at 14,500 g. (Each 7.5 ml sample was split among five 2 ml tubes.) After removing the supernatant, 150 µl of Trizol (Invitrogen) was added to each pellet.  The five 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 50 µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2 µl DNase I (Ambion, 2 U/ul) 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 centrifugation (10 minutes, 4°C,  14,500 g).  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 kept at room temperature for 30 minutes or for 4 hours at 4°C.  RNA was stored at -80°C.  RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer.

source_name: LS3767 (CB15 del pleC)

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 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.  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 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.

extract_protocol: For RNA extraction, ~7.5 ml of cell culture was pelleted by centrifugation for 1 minute in a microfuge at 14,500 g. (Each 7.5 ml sample was split among five 2 ml tubes.) After removing the supernatant, 150 µl of Trizol (Invitrogen) was added to each pellet.  The five 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 50 µl Nuclease free water (Ambion). To digest DNA in the sample, we added 2ul DNase I (Ambion, 2U/ul) 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 20ul 3M sodium acetate (pH 5.2) and 500 µl -20°C ethanol and freezing overnight at -80°C.  RNA was then pelleted by centrifugation (10 minutes, 4°C,  14,500 g).   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 kept at room temperature for 30 minutes or for 4 hours at 4°C.  RNA was stored at -80°C.  RNA quality was visualized on agarose gels and concentration was measured using a spectrophotometer.

tag: Asynchronous exponential phase culture of LS3767 (CB15 del pleC) grown in PYE at 30°C to OD600 = 0.2717.


title: Synechocystis cells treated 300 min with 50 µM cadmium sulfate, replicate 4

status: Public on Dec 07 2005

city: Gif sur yvette CEDEX

first: Franck

last: Chauvat

department: DSV/DBJC

country: France

institute: CEA Saclay

scan_protocol: Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000B fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.

data_processing: After background correction and removal of flagged values, expression ratios were normalised using LOWESS method (Genespring software) to obtain values given in the data table.

organism: Synechocystis sp. PCC 6803

source_name: Synechocystis cells treated 300 min with 0 µM cadmium sulfate

label_protocol: The cDNA from treated and untreated cells RNA was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50 mM sodium bicarbonate, pH 9.0, followed by quenching with 4 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 30 µl of buffer containing 21% formamide, 0.2% DSD, 1x denhardt's, 7x SSPE, and 10 µg each of mouse Cot1 DNA, yeast tRNA and polydeoxyadenylic acid (to limit non-specific hybridization) for hybridization to the microarray.

growth_protocol: 300 ml of mid-log phase liquid culture grown in standard conditions (2.5x107 cells ml-1) were rapidly concentrated 40-fold by centrifugation and spotted as 20 µl dots on BG11 plates with or without Cadmium sulfate, 50 µM. Then the plates were incubated ˆ 30¡C under standard light for 300 min. Following growth, all cell samples were rapidly harvested and disrupted.

extract_protocol: RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 µg) was annealed with 2 µg of random hexanucleotide primers and 0.5 ng of Human TFR mRNA (Takara) used as external control. Then, the reverse-transcription into cDNA was done with Superscript II reverse transcriptase for 2h at 42¡C in the presence of 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.1 mM dTTP, 0.4 mM amino-allyl dUTP. After hydrolysis of RNA with 3 U of RNAse H (Amersham Pharmacia), cDNA was purified with a Microcon-30 concentrator.


title: Wild type pancreatic islets_replicate 2 (MG-U74B)

description: Pancreatic islets total RNA


title: slr1738 null mutant cadmium (CdSO4 50 (M 180 min), replicate 2

description: (Slr1738 mutant treated 180 min with CdSO4 50 (M

source_name: wild type strain treated 180 min with CdSO4 50 (M

growth_protocol: 300 ml of mid-log phase liquid culture grown in standard conditions (2.5x107 cells ml-1) were rapidly concentrated 40-fold by centrifugation and spotted as 20 µl dots on BG11 plates with CdSO4 (50 (M). Then the plates were incubated ˆ 30¡C under standard light for 180 min. Following growth, all cell samples were rapidly harvested and disrupted.


title: podJ_deletion-rep1

tag: Asynchronous exponential phase culture of CB15 (LS3032) grown in PYE at 30°C to OD600 = 0.2526.

tag: Asynchronous exponential phase culture of LS3766 (CB15 del podJ) grown in PYE at 30°C to OD600 = 0.3059.


title: Synechocystis cells treated 300 min with 50 µM cadmium sulfate, replicate 3

tag: Synechocystis cells treated 300 min with 0 µM cadmium sulfate


title: pleC_deletion-rep4

source_name: LS3768 (CB15 del pleC)

tag: Asynchronous exponential phase culture of LS3768 (CB15 del pleC) grown in PYE at 30°C to OD600 = 0.2676.