title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 32 hours (NCBI build 35)

city: Santa Clara

first: Thomas

middle: R

last: Gingeras

address: 3380 Central Expressway

institute: Affymetrix, Inc.

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer's protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer's protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, PolyA+ RNA, HL-60 Cells, 32 hours Retinoic Acid

molecule: polyA RNA


title: MUR40325A

status: Public on Dec 08 2005

type: RNA

city: Berkely

first: Sanchita

last: Bhattacharya

state: CA

address: 1 Cyclotron Rd

country: USA

institute: MIT

description:
Oct-1 mutant_Radiated
Replicate 4

molecule: total RNA

organism: Mus musculus

source_name: Early passage primary MEFs


title: PolyA+ RNA from cytosol of HeLa Cells (NCBI build 35)

status: Public on Dec 13 2005

description:
HeLa adherent cells were grown in Dulbecco's Modified Eagle's Medium (DMEM); supplemented with 10% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to confluency (medium and serum were puraches from HyClone; supplemental components were purchased from Invitrogen).
Cytosolic RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer's protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer's protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, Cytosolic PolyA+ RNA, HeLa Cells

molecule: cytoplasmic RNA

organism: Homo sapiens

source_name: PolyA+ RNA from cytosol of HeLa Cells


title: WT40204A

description:
Wild Type
Replicate 2


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 0 hours (NCBI build 35)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer's protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer's protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, PolyA+ RNA, HL-60 Cells, 0 hours Retinoic Acid


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 2 hours (NCBI build 35)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer's protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer's protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, PolyA+ RNA, HL-60 Cells, 2 hours Retinoic Acid


title: MUR40323A

description:
Oct-1 mutant_Radiated
Replicate 3


title: MUR40203A

description:
Oct-1 mutant_Radiated
Replicate 1


title: H3K9K14D ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBI build 35) - strict analysis parameters

type: genomic

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against H3K9K14D overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, H3K9K14D, HL60 Cells, 0 hours Retinoic Acid

molecule: genomic DNA

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 0 hours


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 32 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against Pol2 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, Pol2, HL60 Cells, 32 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 32 hours


title: MU40202A

description: Oct-1 mutant


title: MU40322A

description:
Oct-1 mutant
Replicate 3


title: MU40324A

description:
Oct-1 mutant
Replicate 4


title: MUR40207A

description:
Oct-1 mutant_Radiated
Replicate 2


title: WTR40319A

description:
Wild type _Radiated
Replicate 3


title: Caulobacter_pye_cell_cycle_90min_rep2

city: Princeton

first: Alison

last: Hottes

state: NJ

address: 220 Carl Icahn Lab Washington Road

department: Lewis-Sigler Institute for Integrative Genomics

institute: Princeton University

description: Sample taken 90 min into a cell cycle in PYE media

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.

organism: Caulobacter vibrioides

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 90 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: WT40200A

description:
Wild Type
Replicate 1


title: Caulobacter_pye_cell_cycle_80min_rep1

description: Sample taken 80 min into a cell cycle in PYE media

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 80 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: Caulobacter_pye_cell_cycle_90min_rep1


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 8 hours (NCBI build 35)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer's protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, PolyA+ RNA, HL-60 Cells, 8 hours Retinoic Acid


title: MU40206A

description:
Oct-1 mutant
Replicate 2


title: H3K9K14D ChIP from Retinoic Acid Stimulated HL60 Cells, 8 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against H3K9K14D overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, H3K9K14D, HL60 Cells, 8 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 8 hours


title: Caulobacter_pye_cell_cycle_80min_rep2


title: p63_ActD ChIP from Retinoic Acid Stimulated ME180 Cells, 0 hours (NCBI build 35) - strict analysis parameters

description:
ME180 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10^6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10^8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10^7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against p63_ActD overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, p63_ActD, ME180 Cells, 0 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated ME180 Cells, 0 hours


title: Caulobacter_pye_cell_cycle_60min_rep1

description: Sample taken 60 min into a cell cycle in PYE media

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 60 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: HisH4 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against HisH4 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, HisH4, HL60 Cells, 2 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 2 hours


title: Caulobacter_pye_cell_cycle_40min_rep1

description: Sample taken 40 min into a cell cycle in PYE media

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 40 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: Caulobacter_pye_cell_cycle_50min_rep2

description: Sample taken 50 min into a cell cycle in PYE media

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 50 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: Caulobacter_pye_cell_cycle_70min_rep1

description: Sample taken 70 min into a cell cycle in PYE media

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 70 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: H3K9K14D ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against H3K9K14D overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, H3K9K14D, HL60 Cells, 2 hours Retinoic Acid


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 8 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against Pol2 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, Pol2, HL60 Cells, 8 hours Retinoic Acid


title: Caulobacter_pye_cell_cycle_30min_rep1

description: Sample taken 30 min into a cell cycle in PYE media

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 30 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: Caulobacter_pye_cell_cycle_20min_rep1

description: Sample taken 20 min into a cell cycle in PYE media

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 20 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: Caulobacter_pye_cell_cycle_50min_rep1


title: Caulobacter_pye_cell_cycle_100min_rep1

description: Sample taken 100 min into a cell cycle in PYE media

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 100 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: HisH4 ChIP from Retinoic Acid Stimulated HL60 Cells, 8 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against HisH4 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, HisH4, HL60 Cells, 8 hours Retinoic Acid


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against Pol2 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, Pol2, HL60 Cells, 0 hours Retinoic Acid


title: PolyA+ RNA from cytosol of GM06990 Cells (NCBI build 35)

description:
GM06990 suspension cells were grown in Rosswel Park Memorial Institute (RPMI) 1640 medium; supplemented with 15% FBS (heat-inactivated), and 1% Penicillin-Streptomycin, at 37C in 5% CO2 (medium and serum were puraches from HyClone; supplemental components were purchased from Invitrogen).
Cytosolic RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer's protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer's protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, Cytosolic PolyA+ RNA, GM06990 Cells

tag: Coriell cell line repository identifier


title: HisH4 ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against HisH4 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, HisH4, HL60 Cells, 0 hours Retinoic Acid


title: H3K9K14D ChIP from Retinoic Acid Stimulated HL60 Cells, 32 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against H3K9K14D overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, H3K9K14D, HL60 Cells, 32 hours Retinoic Acid


title: HisH4 ChIP from Retinoic Acid Stimulated HL60 Cells, 32 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against HisH4 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, HisH4, HL60 Cells, 32 hours Retinoic Acid


title: Caulobacter_m2g_cell_cycle_90min_rep1

description: Sample taken 90 min into a cell cycle in M2G 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  45 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 M2G at 30°C to OD660 = 0.4877.

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  45 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 M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 90 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_60min_rep2

description: Sample taken 60 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 60 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBI build 35) - strict analysis parameters

description:
HL60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10_6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10_8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10_7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against Pol2 overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, Pol2, HL60 Cells, 2 hours Retinoic Acid


title: Caulobacter_m2g_cell_cycle_45min_rep2

description: Sample taken 45 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 45 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_75min_rep2

description: Sample taken 75 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 75 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_60min_rep1


title: Caulobacter_m2g_cell_cycle_30min_rep2

description: Sample taken 30 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 30 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_45min_rep1


title: Caulobacter_m2g_cell_cycle_15min_rep2

description: Sample taken 15 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 15 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_30min_rep1


title: Caulobacter_m2g_cell_cycle_15min_rep1


title: p63_mActD ChIP from Retinoic Acid Stimulated ME180 Cells, 0 hours (NCBI build 35) - strict analysis parameters

description:
ME180 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10^6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10^8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10^7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against p63_mActD overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel.
The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips  (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Homo sapiens, p63_mActD, ME180 Cells, 0 hours Retinoic Acid


title: Caulobacter_m2g_cell_cycle_135min_rep2

description: Sample taken 135 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 135 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_105min_rep1

description: Sample taken 105 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 105 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_150min_rep1

description: Sample taken 150 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 150 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_135min_rep1


title: Caulobacter_m2g_cell_cycle_120min_rep1

description: Sample taken 120 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 120 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_120min_rep2


title: Caulobacter_m2g_cell_cycle_105min_rep2


title: Caulobacter_m2g_cell_cycle_150min_rep2


title: Caulobacter_m2g_cell_cycle_90min_rep2


title: Caulobacter_m2g_cell_cycle_0min_rep2

description: Sample taken 0 min into a cell cycle in M2G media

tag: Swarmer-stage cells were isolated from a 30°C log phase M2G culture and released into M2G at 30°C.  Samples were taken every 15 minutes.  This sample was taken at 0 min. At 0 minutes, the OD660 was 0.43; at 150 minutes, the OD660 was 0.80.


title: Caulobacter_m2g_cell_cycle_0min_rep1


title: Caulobacter_m2g_cell_cycle_75min_rep1


title: DivK-cs cell cycle-5h-rep1

description: Sample taken 5h into the DivK-cs modified cell cycle

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 5h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: Caulobacter_pye_cell_cycle_10min_rep1

description: Sample taken 10 min into a cell cycle in PYE media

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

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 10 min. At 0 minutes, the OD660 was 0.48; at 150 minutes, the OD660 was 0.96


title: DivK-cs cell cycle-5h-rep2


title: DivK-cs cell cycle-4h-rep1

description: Sample taken 4h 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 4h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-370m-rep2

description: Sample taken 370min 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 370min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-340m-rep2

description: Sample taken 340min 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 340min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-320m-rep1

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: DivK-cs cell cycle-370m-rep1


title: DivK-cs cell cycle-310m-rep1

description: Sample taken 310min 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 310min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-350m-rep2

description: Sample taken 350min 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 350min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-380m-rep1

description: Sample taken 380min 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 380min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-3h-rep1

description: Sample taken 3h 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 3h. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-350m-rep1


title: DivK-cs cell cycle-340m-rep1


title: DivK-cs cell cycle-330m-rep1

description: Sample taken 330min 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 330min. At 0 minutes, the OD660 was 0.42; at 380 minutes, the OD660 was ~1.


title: DivK-cs cell cycle-310m-rep2