title: Native RPE #2

description:
Description
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions
3.	How many hybridizations in exp: 20
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE
6.	Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors. Sample #2 is from an 80 year old WM with a death to enucleation time of 3 hours, 35 min.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

source_name: Native RPE #2


title: 79a-21dpi-4

status: Public on Oct 13 2004

type: RNA

city: Winnipeg

first: Garrett

last: Sorensen

country: Canada

institute: Canadian Science Centre for Human and Animal Health

description:
C57BL/6 mice were inoculated intracerebrally with 20 µl of 1% brain homogenate which was obtained from the TSE Resource Centre, Institute for Animal Health, Compton, UK.  Mock-infected control mice were inoculated intra-cerebrally with 20 µl of PBS.  The brains were removed at the appropriate time-points and stored at -80 C in RNALater (Ambion) until all experimental time points were completed.
Total RNA was isolated from the stored tissue samples by homogenization in Trizol reagent (Invitrogen). RNA was further purified using RNeasy columns (Qiagen) according to the manufacturers instructions. 10 µg of total RNA as a template for oligo -dT primed reverse transcription and incorporated amino allyl -dUTP (Sigma) into the cDNA, essentially following the Institute for Genomic Research, Rockville, MD, USA (TIGR) published protocol http://pga.tigr.org/sop/M004_1a.pdf
The remaining RNA template was hydrolysed and the cDNA product purified using the QIAquick PCR purification system (Qiagen). cDNA was dried down, then resuspended in 0.1 M Na2CO3, pH9.0 buffer, and incubated for 1 hour in either an Alexa Fluor555 (AF555) or Alexa Fluor647(AF647) mono-functional reactive dye (Molecular Probes) made up in DMSO.  Labelled cDNA was also purified using QIAquick PCR purification columns.
Labelled cDNA was dried down and resuspended in 60 µl DIG Easy Hyb hybridisation buffer (Roche) containing calf thymus DNA (Sigma) and yeast tRNA (Life Technologies). The labelled cDNAs were applied to arrays under M Series Lifter Slips (Erie Scientific Company) and the slides incubated in a hybridisation chamber at 42oC for 16 hours. Following hybridisation lifter slips were removed and the arrays washed a total of 3 times for 5 minutes first in1 X SSC, 0.2% SDS, and then 3 times for 5 minutes in 0.1 X SSC, 0.2% SDS pre-warmed to 42°C. A final wash in 0.1 X SSC at room temperature was performed, and slides centrifuged to dry. Slides were immediately scanned using a VersArray scanner (BioRad) at appropriate laser power and PhotoMultiplier Tube settings so that high intensity spots were not saturated. Spots were quantitated and background signals removed using ArrayPro software (Media Cybernetics, Inc).

molecule: total RNA

organism: Mus musculus

source_name: Mock infected, 21 d.p.i.


title: ME7-end-7

source_name: Mock infected, endpoint


title: Differentiated #3, serum withdrawn

city: Baltimore

first: James

middle: T

last: Handa

state: MD

address: 600 N. Wolfe St.

department: Wilmer Eye Institute

country: USA

institute: Johns Hopkins Medical Institutes

description:
Description
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions
3.	How many hybridizations in exp: 20
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE
6.	Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

organism: Homo sapiens

source_name: Differentiated #3, serum withdrawn


title: Differentiated RPE, in serum #3

source_name: Differentiated RPE, in serum #3


title: Differentiated RPE, in serum #1

source_name: Differentiated RPE, in serum #1


title: 79a-end-1


title: 79a-21dpi-1


title: 79a-21dpi-5


title: ME7-21dpi-7


title: Differentiated RPE, serum withdrawn #2

source_name: Differentiated RPE, serum withdrawn #2


title: Differentiated RPE, serum withdrawn #1

source_name: Differentiated RPE, serum withdrawn #1


title: Differentiated RPE, in serum #2

source_name: Differentiated RPE, in serum #2


title: ME7-21dpi-8


title: Confluent RPE, in serum #2

source_name: Confluent RPE, in serum #2


title: ME7-21dpi-5


title: Confluent RPE, in serum #3

source_name: Confluent RPE, in serum #3


title: ME7-21dpi-6


title: ME7-21dpi-4


title: Confluent RPE, serum withdrawn #2

source_name: Confluent RPE, serum withdrawn #2


title: ME7-100dpi-2

source_name: Mock infected, 100 d.p.i.


title: ME7-21dpi-3


title: ME7-21dpi-1


title: Confluent RPE, serum withdrawn #3

source_name: Confluent RPE, serum withdrawn #3


title: ME7-100dpi-1


title: Confluent RPE, serum withdrawn #1

source_name: Confluent RPE, serum withdrawn #1


title: ME7-21dpi-2


title: 79a-21dpi-3


title: 79a-21dpi-2


title: Rat SCN Sample 16 (Technical replicate - 42 hr)

status: Public on Oct 09 2004

city: College Station

first: Gus

middle: John

last: Menger

state: TX

department: Anatomy and Neurobiology

institute: Texas A&M Health Science Center

description: To determine whether immortalized cells derived from the rat SCN (SCN 2.2) retain intrinsic rhythm-generating properties characteristic of the SCN, oscillatory properties of the SCN2.2 transcriptome were analyzed and compared to those found in the rat SCN in vivo using rat U34A Affymetrix GeneChips.  In this comparison, adult male Long-Evans rats (175-200g; N=45) were housed under a standard 12h light:12h dark photoperiod (LD 12:12; lights-on at 0600 hr). At 1800 hr (circadian time [CT] 12), animals were exposed to constant darkness (DD) and 12 hours later (0600 hr or CT 0), sacrificed under isoflurane anesthesia at 6-hr intervals (N=5) for 48 hours by decapitation using an infrared viewer.  After the eyes were removed in the dark, SCN tissue was immediately dissected under dim light, frozen in liquid nitrogen, and stored at –80C. SCN tissue from individual animals was separately homogenized in TRIzol reagent by aspiration through a 25-gauge needle and then extracted cellular RNA for all animals at each timepoint was pooled into a single sample. RNA samples were subjected to on-column treatment with DNAse-1 to digest genomic DNA and then were stored at –80°C before routine processing for GeneChip analysis.

organism: Rattus norvegicus

source_name: Long-Evans Rat Suprachiasmatic Nuclei


title: Rat SCN Sample 15 (Technical replicate - 36 hr)


title: Rat SCN Sample 13 (Technical replicate - 24 hr)


title: RD02D010

status: Public on Oct 12 2004

city: Houston

first: Zhiyong

last: Yang

institute: Baylor College of Medicine

description:
Whole heads of Rh1-GAL4/P{UAS-dPABP-FLAG} flies were seperated from bodies using liquid nitrogen freezing and sieve (0.710 mm) seperation. About 200 fly heads were fixed in 1 ml of 1xPBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) containing 1% formaldehyde and 0.5% NP40 for 30 min at 4¡æ. After fixation, 140 ul of 2 M glycine was added and the sample incubated at 4¡æ for 5 min. The heads were rinsed 3 times with 1xPBS and homogenized in 0.8 ml of homogenization buffer. To prepare homogenization buffer (HB), a solution of 150 mM NaCl, 50 mM Hepes buffer at pH 7.6, 1 mM EGTA, 15 mM EDTA, and 10% glycerol was treated with diethyl pyrocarbonate. Immediately before use, vanadyl ribonucleoside complex (Sigma), SUPERase-In (Ambion), and a protease inhibitor cocktail tablet (Roche) were added to the solution to final concentrations of 8 mM, 50 U/ml and 1 tablet/50 ml, respectively. The homogenate was sonicated and cleared by centrifugation at 13,000 xg for 10 min. RNA bound to the FLAG-tagged PABP was recovered by co-immunoprecipitation using anti-FLAG-M2 affinity agarose beads (Sigma). Before use, the agarose beads were washed four times with HB at 4¡æ. For co-immunoprecipitation, we gently mixed 0.8 ml of homogenate with anti-FLAG-M2 affinity agarose beads from 100 ul of bead suspension for 2 hr at 4¡æ. The beads were then washed four times with HB at 4¡æ. The RNA::PABP crosslink was then reversed by incubating the beads in 100 ul of elution buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1.3% SDS, 50 U/ml SUPERase-In) at 65¡æ for 30 min. To isolate RNA, 100 ul of eluant was mixed with 400 ul of Trizol (Invitrogen) and then mixed with 100 ul of chloroform. The mixture was incubated at room temperature for 10 min and then centrifuged at 12,000 xg for 10 min at 4¡æ. The aqueous supernatant was transferred to a new tube and mixed with 250 ul of isopropanol, chilled at ¨C80¡æ overnight, and centrifuged at 12,000 xg for 10 min at 4¡æ. The RNA pellet was rinsed once with 0.5 ml of 70% ethanol and then dissolved in 20 ul of RNase free water. Targets were produced using standard Affymetrix procedures from all mRNA sample recovered. 5 ug cRNA was generated and used for hybridization.
This sample is photorecetor cells-enriched sample 2 from Rh1-GAL4/P{UAS-dPABP-FLAG} flies.
Keywords = Drosophila, mRNA tagging, photoreceptor cell-specific genes

organism: Drosophila melanogaster

source_name: photoreceptor cells R1-R6


title: Rat SCN Sample 10 (Technical replicate - 6 hr)


title: Rat SCN Sample 9 (Technical replicate - 0 hr)


title: Rat SCN Sample 7b (Exp 1 - 36 hr b)


title: Rat SCN Sample 11 (Technical replicate - 12 hr)


title: RD02D009R

description:
Whole heads of Rh1-GAL4/P{UAS-dPABP-FLAG} flies were seperated from bodies using liquid nitrogen freezing and sieve (0.710 mm) seperation. About 200 fly heads were fixed in 1 ml of 1xPBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) containing 1% formaldehyde and 0.5% NP40 for 30 min at 4¡æ. After fixation, 140 ul of 2 M glycine was added and the sample incubated at 4¡æ for 5 min. The heads were rinsed 3 times with 1xPBS and homogenized in 0.8 ml of homogenization buffer. To prepare homogenization buffer (HB), a solution of 150 mM NaCl, 50 mM Hepes buffer at pH 7.6, 1 mM EGTA, 15 mM EDTA, and 10% glycerol was treated with diethyl pyrocarbonate. Immediately before use, vanadyl ribonucleoside complex (Sigma), SUPERase-In (Ambion), and a protease inhibitor cocktail tablet (Roche) were added to the solution to final concentrations of 8 mM, 50 U/ml and 1 tablet/50 ml, respectively. The homogenate was sonicated and cleared by centrifugation at 13,000 xg for 10 min. RNA bound to the FLAG-tagged PABP was recovered by co-immunoprecipitation using anti-FLAG-M2 affinity agarose beads (Sigma). Before use, the agarose beads were washed four times with HB at 4¡æ. For co-immunoprecipitation, we gently mixed 0.8 ml of homogenate with anti-FLAG-M2 affinity agarose beads from 100 ul of bead suspension for 2 hr at 4¡æ. The beads were then washed four times with HB at 4¡æ. The RNA::PABP crosslink was then reversed by incubating the beads in 100 ul of elution buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1.3% SDS, 50 U/ml SUPERase-In) at 65¡æ for 30 min. To isolate RNA, 100 ul of eluant was mixed with 400 ul of Trizol (Invitrogen) and then mixed with 100 ul of chloroform. The mixture was incubated at room temperature for 10 min and then centrifuged at 12,000 xg for 10 min at 4¡æ. The aqueous supernatant was transferred to a new tube and mixed with 250 ul of isopropanol, chilled at ¨C80¡æ overnight, and centrifuged at 12,000 xg for 10 min at 4¡æ. The RNA pellet was rinsed once with 0.5 ml of 70% ethanol and then dissolved in 20 ul of RNase free water. Targets were produced using standard Affymetrix procedures from all mRNA sample recovered. 10 ug cRNA was generated and used for hybridization.
This sample is photorecetor cells-enriched sample 1 from Rh1-GAL4/P{UAS-dPABP-FLAG} flies.
Keywords = Drosophila, mRNA tagging, photoreceptor cell-specific genes


title: Rat SCN Sample 12 (Technical replicate - 18 hr)


title: Rat SCN Sample 6 (Exp 1 - 30 hr)


title: Rat SCN Sample 5b (Exp 1 - 24 hr b)


title: Rat SCN Sample 7a (Exp 1 - 36 hr a)


title: Rat SCN Sample 8 (Exp 1 - 42 hr)


title: RD02D013

description:
Whole heads of Rh1-GAL4/P{UAS-dPABP-FLAG} flies were seperated from bodies using liquid nitrogen freezing and sieve (0.710 mm) seperation. About 200 fly heads were fixed in 1 ml of 1xPBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) containing 1% formaldehyde and 0.5% NP40 for 30 min at 4¡æ. After fixation, 140 ul of 2 M glycine was added and the sample incubated at 4¡æ for 5 min. The heads were rinsed 3 times with 1xPBS and homogenized in 0.8 ml of homogenization buffer. To prepare homogenization buffer (HB), a solution of 150 mM NaCl, 50 mM Hepes buffer at pH 7.6, 1 mM EGTA, 15 mM EDTA, and 10% glycerol was treated with diethyl pyrocarbonate. Immediately before use, vanadyl ribonucleoside complex (Sigma), SUPERase-In (Ambion), and a protease inhibitor cocktail tablet (Roche) were added to the solution to final concentrations of 8 mM, 50 U/ml and 1 tablet/50 ml, respectively. The homogenate was sonicated and cleared by centrifugation at 13,000 xg for 10 min. RNA bound to the FLAG-tagged PABP was recovered by co-immunoprecipitation using anti-FLAG-M2 affinity agarose beads (Sigma). Before use, the agarose beads were washed four times with HB at 4¡æ. For co-immunoprecipitation, we gently mixed 0.8 ml of homogenate with anti-FLAG-M2 affinity agarose beads from 100 ul of bead suspension for 2 hr at 4¡æ. The beads were then washed four times with HB at 4¡æ. The RNA::PABP crosslink was then reversed by incubating the beads in 100 ul of elution buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1.3% SDS, 50 U/ml SUPERase-In) at 65¡æ for 30 min. To isolate RNA, 100 ul of eluant was mixed with 400 ul of Trizol (Invitrogen) and then mixed with 100 ul of chloroform. The mixture was incubated at room temperature for 10 min and then centrifuged at 12,000 xg for 10 min at 4¡æ. The aqueous supernatant was transferred to a new tube and mixed with 250 ul of isopropanol, chilled at ¨C80¡æ overnight, and centrifuged at 12,000 xg for 10 min at 4¡æ. The RNA pellet was rinsed once with 0.5 ml of 70% ethanol and then dissolved in 20 ul of RNase free water. Targets were produced using standard Affymetrix procedures from all mRNA sample recovered. 4.7 ug cRNA was generated and used for hybridization.
This sample is photorecetor cells-enriched sample 4 from Rh1-GAL4/P{UAS-dPABP-FLAG} flies.
Keywords = Drosophila, mRNA tagging, photoreceptor cell-specific genes


title: Rat SCN Sample 5a (Exp 1 - 24 hr a)


title: Rat SCN Sample 2 (Exp 1 - 6 hr)

description: To determine whether immortalized cells derived from the rat SCN (SCN 2.2) retain intrinsic rhythm-generating properties characteristic of the SCN, oscillatory properties of the SCN2.2 transcriptome were analyzed and compared to those found in the rat SCN in vivo using rat U34A Affymetrix GeneChips.  In this comparison, adult male Long-Evans rats (175-200g; N=45) were housed under a standard 12h light:12h dark photoperiod (LD 12:12; lights-on at 0600 hr). At 1800 hr (circadian time [CT] 12), animals were exposed to constant darkness (DD) and 12 hours later (0600 hr or CT 0), sacrificed under isoflurane anesthesia at 6-hr intervals (N=5) for 48 hours by decapitation using an infrared viewer.  After the eyes were removed in the dark, SCN tissue was immediately dissected under dim light, frozen in liquid nitrogen, and stored at –800C. SCN tissue from individual animals was separately homogenized in TRIzol reagent by aspiration through a 25-gauge needle and then extracted cellular RNA for all animals at each timepoint was pooled into a single sample. RNA samples were subjected to on-column treatment with DNAse-1 to digest genomic DNA and then were stored at –80°C before routine processing for GeneChip analysis.


title: Rat SCN Sample 4 (Exp 1 - 18 hr)


title: Rat SCN Sample 1 (Exp 1 - 0 hr)


title: Rat SCN Sample 3 (Exp 1 - 12 hr)


title: FRD875 4

status: Public on Oct 08 2004

city: Winston-Salem

first: Josyf

last: Mychaleckyj

state: NC

department: Center for Human Genomics

institute: Wake Forest Univ School of Medicine

description:
Calu-3 human lung epithelial cells were seeded onto 6-well plates and grown to about 80% confluency. Epithelial monolayers were infected with 10**8 CFUs per ml of one of the bacterial strains (FRD1, FRD440, FRD875, or FRD1234) for 60 min and then washed extensively with phosphate-buffered saline. The cultures were subsequently incubated in fresh RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. After 6 h, total RNA from the cells was prepared with RNAzol and further purified with RNEasy (Qiagen) for microarray hybridization. Control samples of Calu-3 cells were treated similarly, but not infected with bacteria. Single array image analysis was performed using MAS 5 (Affymetrix) to derive probeset intensities and detection p-values. All arrays were globally scaled to 500.
Keywords = CF, Cystic Fibrosis, Pseudomonas, Alginate, Flagellin

source_name: Calu-3 human lung epithelial cells


title: FRD440 4


title: RD02D011

description:
Whole heads of Rh1-GAL4/P{UAS-dPABP-FLAG} flies were seperated from bodies using liquid nitrogen freezing and sieve (0.710 mm) seperation. About 200 fly heads were fixed in 1 ml of 1xPBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl) containing 1% formaldehyde and 0.5% NP40 for 30 min at 4¡æ. After fixation, 140 ul of 2 M glycine was added and the sample incubated at 4¡æ for 5 min. The heads were rinsed 3 times with 1xPBS and homogenized in 0.8 ml of homogenization buffer. To prepare homogenization buffer (HB), a solution of 150 mM NaCl, 50 mM Hepes buffer at pH 7.6, 1 mM EGTA, 15 mM EDTA, and 10% glycerol was treated with diethyl pyrocarbonate. Immediately before use, vanadyl ribonucleoside complex (Sigma), SUPERase-In (Ambion), and a protease inhibitor cocktail tablet (Roche) were added to the solution to final concentrations of 8 mM, 50 U/ml and 1 tablet/50 ml, respectively. The homogenate was sonicated and cleared by centrifugation at 13,000 xg for 10 min. RNA bound to the FLAG-tagged PABP was recovered by co-immunoprecipitation using anti-FLAG-M2 affinity agarose beads (Sigma). Before use, the agarose beads were washed four times with HB at 4¡æ. For co-immunoprecipitation, we gently mixed 0.8 ml of homogenate with anti-FLAG-M2 affinity agarose beads from 100 ul of bead suspension for 2 hr at 4¡æ. The beads were then washed four times with HB at 4¡æ. The RNA::PABP crosslink was then reversed by incubating the beads in 100 ul of elution buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1.3% SDS, 50 U/ml SUPERase-In) at 65¡æ for 30 min. To isolate RNA, 100 ul of eluant was mixed with 400 ul of Trizol (Invitrogen) and then mixed with 100 ul of chloroform. The mixture was incubated at room temperature for 10 min and then centrifuged at 12,000 xg for 10 min at 4¡æ. The aqueous supernatant was transferred to a new tube and mixed with 250 ul of isopropanol, chilled at ¨C80¡æ overnight, and centrifuged at 12,000 xg for 10 min at 4¡æ. The RNA pellet was rinsed once with 0.5 ml of 70% ethanol and then dissolved in 20 ul of RNase free water. Targets were produced using standard Affymetrix procedures from all mRNA sample recovered. 10 ug cRNA was generated and used for hybridization.
This sample is photorecetor cells-enriched sample 3 from Rh1-GAL4/P{UAS-dPABP-FLAG} flies.
Keywords = Drosophila, mRNA tagging, photoreceptor cell-specific genes


title: FRD875 2


title: FRD440 2


title: FRD875 3


title: FRD875 1


title: FRD1234 3


title: FRD1 2


title: CONTROL 1


title: FRD440 1


title: FRD1 3


title: Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals (HS_11662)

status: Public on Oct 29 2004

city: New Haven

first: Paul

last: Bertone

state: CT

institute: Yale University

description:
Triple-selected poly(A)+ human liver tissue RNA pooled from several individuals was obtained from Ambion (Austin, TX). First-strand cDNA was generated from 4 µg RNA/rxn with (RNase H-) M-MLV reverse transcriptase, primed with equimolar concentrations of oligo(dT18) and random decamers. The reactions were carried out at 42°C for 2 hours in the presence of 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.15 mM dTTP and 0.15 mM amino-allyl dUTP to facilitate the secondary labeling of an amine-reactive fluorescent conjugate.
Following reverse transcription the products were heated at 95°C for 5 minutes to denature the RNA:DNA hybrids and heat-inactivate the reverse transcriptase, after which the RNA template was hydrolyzed by incubating in 0.2 M NaOH at 65°C for 15 minutes. The reaction was then neutralized in 0.3 M HEPES (pH 7.0). Reverse transcription products from 20 separate reactions were produced in this manner and pooled to reduce technical variability between samples.
The cDNAs were precipitated in ethanol:isopropanol (1:1 v/v) and resuspended in 0.1 M NaHCO3 to facilitate coupling of Alexa Fluor 555 NHS esters (Molecular Probes, Eugene, OR) to the reactive groups of the amino-allyl dUTPs. Following incubation at room temperature for 1 hour, the labeled cDNAs were purified with CyScribe GFX glass fiber spin columns (Amersham Bioscience, Piscataway, NJ) and isopropanol-precipitated.
Microarrays were hybridized with 2-3 µg labeled cDNA in 320 µL hybridization buffer (50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20) for 20 hours at 50°C. Hybridizations were performed in disposable adhesive chambers (Grace BioLabs, Bend, OR) in a hybridization oven with constant agitation.
After hybridization, the arrays were washed on an orbital platform in non-stringent buffer (6× SSPE, 0.01% [v/v] Tween-20) for 10 minutes at room temperature, then in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween-20) for 30 minutes at 45°C. This was followed by a 5-minute wash in non-stringent buffer and a 4-minute wash in 0.2X SSC.
The arrays were dried with compressed nitrogen. Fluorescence micrographs were acquired with an Axon 4000B laser scanner at 5 µm resolution and intensity data were extracted with NimbleScan software (NimbleGen Systems).
Keywords = liver tissue, poly(A)+ RNA

source_name: Alexa555-labeled cDNA derived from human liver poly(A)+ RNA


title: FRD1234 2


title: CONTROL 3


title: CONTROL 4


title: FRD1234 1


title: CONTROL 2


title: FRD1 4


title: FRD440 3


title: Rat SCN Sample 14 (Technical replicate - 30 hr)


title: chlorpromazine-treated mouse brain

status: Public on Sep 24 2004

city: Kagoshima

first: Shin-ichi

last: Iwata

state: Kagoshima

address: Sakuragaoka 8-35-1

department: Pharmacology

country: Japan

institute: Kagoshima University

description:
Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Dose of 25 mg/kg chlorpromazine HCl (Sigma-Aldrich Co., St. Louis, MO)was used in the study. The drug was dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight.
Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit as per the manufacturer?s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80?C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5. Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kit?s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60?C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6דSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1דSC with 0.005% Triton X-102 at 4?C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel.

Keywords = chlorpromazine
Lot batch = US22502606_16011978017254

source_name: vehicle-treated mouse brain

source_name: chlorpromazine-treated mouse brain


title: quetiapine-treated mouse brain

description:
Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Dose of 18.75 mg/kg quetiapine fumarate (gift from AstraZeneca, Macclesfield, UK) was used in the study. The drug was dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight.
Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit as per the manufacturer’s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80°C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5. Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kit’s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60°C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6×SSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1×SSC with 0.005% Triton X-102 at 4°C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel.

Keywords = quetiapine
Lot batch = US22502606_16011978017255

source_name: quetiapine-treated mouse brain


title: FRD1 1


title: No added copper sample 3

status: Public on Sep 22 2004

city: Birmingham

first: Chrystala

last: Constantinidou

department: School of Biosciences

country: United Kingdom

institute: University of Birmingham

description: “Pool” RNA was isolated from five independently grown 50ml DMA cultures of E. coli K-12 MG1655. “Test” RNA was isolated from three independently grown 50ml DMA (Defined Medium A) cultures of E. coli K-12 MG1655, for each growth condition (no added CuSO4, or supplemented with either 0.75mM or 2mM copper glycine).  A sample of each culture was taken at mid to late exponential growth phase at OD600 0.8 (measured using a Pharmacia Biotech Ultrospec 2000 in a cuvette with a path length of 10mm).  RNA isolation was performed using the Qiagen RNAprotectTM, RNeasy Mini Kit and RNase-free DNase sets according to the manufacturers’ instructions (Qiagen Ltd, Surrey, UK).  The total RNA isolated from the cultures was quantified using an Agilent Bioanalyser 2100 RNA NanoLabchip, where, the quality of the RNA was indicated from the ratio of absorbance of 23S to 16S ribosomal RNA which was between 1.6 and 2.

organism: Escherichia coli

source_name: DMA no added copper (test)

source_name: DMA no added copper


title: thioridazine-treated mouse brain

description:
Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Dose of 25 mg/kg thioridazine HCl (Sigma-Aldrich Co) was used in the study. The drug was dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight.
Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit as per the manufacturer’s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80°C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5. Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kit’s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60°C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6×SSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1×SSC with 0.005% Triton X-102 at 4°C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel.

Keywords = thioridazine
Lot batch = US22502606_16011978017256

source_name: thioridazine-treated mouse brain


title: olanzapine-treated mouse brain

description:
Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Dose of 1.25 mg/kg olanzapine (gift from Eli Lilly and Company, Indianapolis, IN) was used in the study. The drug was dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight.
Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit as per the manufacturer’s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80°C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5. Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kit’s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60°C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6×SSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1×SSC with 0.005% Triton X-102 at 4°C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel.

Keywords = olanzapine
Lot batch = US22502606_16011978017320

source_name: olanzapine-treated mouse brain


title: No added copper sample 1


title: 2mM Copper sample 3

source_name: DMA + 2mM copper


title: 0.75mM Copper sample 1

source_name: DMA + 0.75mM copper


title: 2mM Copper sample 2


title: No added copper sample 2


title: 0.75mM Copper sample 3


title: PKRwt5+(MB)

city: Montreal

first: Rob

last: Sladek

state: PQ

address: 740 avenue du Docteur Penfield

institute: Montreal Genome Centre

description: NIH3T3 cells expressing wtPKR (NIH3T3 - PKR) under a tetracycline repressed promoter. Cells were grown in DMEM supplemented with 5% FCS in the presence of tetracycline (1 µg/ml).

source_name: Inducible NIH3T3 stable cell line


title: PKRwt5+


title: m577df50 Sample 3

status: Public on Sep 14 2004

city: London

first: Joshua

middle: J.

last: McElwee

address: Gower Street

department: Biology

institute: University College London

description:
Sample is also known as M577mgdf50 3
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging

organism: Caenorhabditis elegans

source_name: Whole animal


title: 2mM Copper sample 1


title: 0.75mM Copper sample 2


title: m577df50 Sample 5

description:
Sample is also known as M577mgdf50 5
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: e1370df50 Sample 3

description:
Sample is also known as E1370mgdf50 3
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577 Sample 3

description:
Sample is also known as m577 6
Genotype is glp-4(bn2); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577df50 Sample 2

description:
Sample is also known as M577mgdf50 2
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577 Sample 2

description:
Sample is also known as m577 5
Genotype is glp-4(bn2); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577df50 Sample 1

description:
Sample is also known as M577mgdf50 1
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577 Sample 4

description:
Sample is also known as m577 7
Genotype is glp-4(bn2); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: e1370df50 Sample 5

description:
Sample is also known as E1370mgdf50 5
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: Sporulation gerR mutant Hour 3.5 exp. 3

status: Public on Sep 01 2004

city: Cambridge

first: Patrick

middle: Yves

last: Eichenberger

state: MA

address: 16 Divinity Avenue

department: Molecular and Cellular Biology

institute: Harvard University

description:
Strains PE454 and SW282 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Three hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE454 and Cy3-dUTP for the RNA sample from strain SW282.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the third repetition of the experiment at this time point.

organism: Bacillus subtilis

source_name: gerR+ (strain PE454)

source_name: gerR mutant (strain SW282)


title: e1370df50 Sample 2

description:
Sample is also known as E1370mgdf50 2
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: e1370 Sample 5

description:
Sample is also known as e1370 6
Genotype is glp-4(bn2); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577df50 Sample 4

description:
Sample is also known as M577mgdf50 4
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: e1370df50 Sample 4

description:
Sample is also known as E1370mgdf50 4
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577 Sample 5

description:
Sample is also known as m577 8
Genotype is glp-4(bn2); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: m577 Sample 1

description:
Sample is also known as m577 3
Genotype is glp-4(bn2); daf-2(m577)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: Sporulation gerR mutant Hour 3.5 exp. 1

description:
Strains PE454 and SW282 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Three hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE454 and Cy3-dUTP for the RNA sample from strain SW282.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the first repetition of the experiment at this time point.


title: e1370 Sample 4

description:
Sample is also known as e1370 5
Genotype is glp-4(bn2); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: e1370 Sample 2

description:
Sample is also known as e1370 3
Genotype is glp-4(bn2); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: Sporulation gerR mutant Hour 3.5 exp. 2

description:
Strains PE454 and SW282 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Three hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE454 and Cy3-dUTP for the RNA sample from strain SW282.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the second repetition of the experiment at this time point.


title: Sporulation sigmaK mutant Hour 4.5 exp. 2

description:
Strains PE455 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Four hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE454 and Cy3-dUTP for the RNA sample from strain PE455.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the second repetition of the experiment at this time point.

source_name: sigmaK+ (PE454)

source_name: sigmaK mutant (PE455)


title: e1370 Sample 3

description:
Sample is also known as e1370 4
Genotype is glp-4(bn2); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: Sporulation sigmaK mutant Hour 4.5 exp. 1

description:
Strains PE455 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Four hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE454 and Cy3-dUTP for the RNA sample from strain PE455.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the first repetition of the experiment at this time point.


title: Sporulation spoIIID mutant Hour 3.5 exp. 2

description:
Strains PE452 and PE456 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Three hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE456 and Cy3-dUTP for the RNA sample from strain PE452.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the second repetition of the experiment at this time point (note the dye swap).

source_name: spoIIID mutant (strain PE456)

source_name: spoIIID+ (strain PE452)


title: Confluent RPE, in serum #1

description:
Description
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions
3.	How many hybridizations in exp: 20
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE
6.	Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

source_name: Confluent RPE, in serum #1


title: e1370 Sample 1

description:
Sample is also known as e1370 2G
Genotype is glp-4(bn2); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging


title: Sporulation spoIIID mutant Hour 3.5 exp. 1

description:
Strains PE452 and PE456 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Three hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE452 and Cy3-dUTP for the RNA sample from strain PE456.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the first repetition of the experiment at this time point.

source_name: spoIIID+ (strain PE456)

source_name: spoIIID mutant (strain PE452)


title: sporulation spoIIID mutant Hour 3.5 exp. 3

description:
Strains PE452 and PE456 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Three hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE452 and Cy3-dUTP for the RNA sample from strain PE456.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the third repetition of the experiment at this time point.


title: Sporulation sigmaK mutant Hour 4.5 exp. 3

description:
Strains PE455 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Four hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PE455 and Cy3-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the third repetition of the experiment at this time point (note the dye swap).

source_name: sigmaK mutant (strain PE455)

source_name: sigmaK+ (strain PE454)


title: Sporulation gerE mutant Hour 5.5 exp. 3

description:
Strains PY79 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Five hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PY79 and Cy3-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the third repetition of the experiment at this time point.

source_name: wild type (PY79 strain)

source_name: gerE mutant (PE454 strain)


title: Sporulation gerE mutant Hour 6.5 exp. 1

description:
Strains PY79 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Six hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PY79 and Cy3-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the first repetition of the experiment at this time point.

source_name: wild type (strain PY79)

source_name: gerE mutant (strain PE454)


title: Sporulation gerE mutant Hour 5.5 exp. 2

description:
Strains PY79 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Five hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy3-dUTP for the RNA sample from strain PY79 and Cy5-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the second repetition of the experiment at this time point (note the dye swap).

source_name: gerE mutant (PE454)

source_name: wild type (PY79)


title: Sporulation gerE mutant Hour 6.5 exp. 2

description:
Strains PY79 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Six hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy3-dUTP for the RNA sample from strain PY79 and Cy5-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the second repetition of the experiment at this time point (note the dye swap).


title: Sporulation gerE mutant Hour 5.5 exp. 1

description:
Strains PY79 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Five hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PY79 and Cy3-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the first repetition of the experiment at this time point.


title: IFN 8a Chip B

city: El Segundo

first: Anton

last: Petrov

state: CA

institute: BioDiscovery Inc

description: At confluence, keratinocytes from the DK-7 cell line were exposed to 100 IU/ml of IFN-g in serum free medium. After 24 hrs, RNA was extracted using the Qiagen Rneasy Mini Kit (Qiagen SA, Cedex, France). All the samples were monitored by agarose gel and with the Agilent 2100 Bioanalyser (Agilent Biotechnologies, Germany) and consistently demonstrated high-quality RNA (28S/18S ratio approximately 2, but always less than 3). According to Affymetrix protocol, 5 µg total RNA was the starting material for all individual samples. In general, total RNA was converted to biotinylated cRNA, hybridized in the Affymetrix probe array cartridge, stained, and then quantified. First and second strand cDNA synthesis was performed using the SuperScript Choice System (Invitrogen AG, Basel, Switzerland), according to manufacturer instructions, but using an oligo-dT primer containing a T7 RNA polymerase binding site. Labeled cRNA was prepared with the RNA Transcript Labeling kit (Enzo Biochem Inc., NY). Biotinylated CTP and UTP were used together with unlabeled NTPs in the reaction, and unincorporated nucleotides were removed with GeneChipâ Cleanup Module (Affymetrix, Inc., Santa Clara, CA) . cRNA (20 µg) was fragmented at 94 °C for 35 min in buffer containing 200 mM Tris-acetate pH 8.1, 500 mM KOAc, and 150 mM MgOAc. Prior to hybridization, fragmented cRNA in hybridization mix ( Buffer containing 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20, 0.5 ng/ml BSA, 0.1 ng/ml herring sperm and Affymetrix controls ), was heated to 95 °C for 5 min, cooled to 45 °C and loaded onto an Affymetrix probe array cartridge. The probe array was incubated for 16 hrs at 45 °C at constant rotation (60 rpm), then exposed to Affymetrix washing and staining protocol. This protocol includes: · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) · one wash with stringent buffer ( 100 mM MES, 0.1 M NaCl, 0.01 % Tween 20 ) · First stain with 0.01 mg/ml streptavidin-phycoerythrin conjugate (Molecular Probes) in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) · Second stain with 3 mg/ml of biotinylated anti-streptavidin + 0.2 mg/ml of IgG in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · Third stain with 0.01 mg/ml streptavidin-phycoerythrin conjugate (Molecular Probes) in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) Probe arrays were scanned at 488 nm using an Argon-ion Laser (made for Affymetrix by Agilent). Readings from the quantitative scanning were analyzed with Affymetrix Gene Expression Analysis Software (MAS 5.0). This experiment was comparing control versus IFN-g treated skin cells (one design factor on two levels). To evaluate the biological and the experimental variability, the following experimental design was planned as shown in Figure 1: "Control" and "IFN-g stimulated" keratinocytes were cultured in triplicate using three individual petri Æ10 culture dishes and RNA was independently extracted from each cultured replicate. For each RNA sample, cRNA synthesis was performed in triplicate and each cRNA pool was hybridized to an Affymetrix U133 Gene Chip.

source_name: keratinocytes from DK-7


title: IFN 8b Chip B


title: IFN 6b Chip B


title: IFN 5c Chip B


title: Sporulation gerE mutant Hour 6.5 exp. 3

description:
Strains PY79 and PE454 were grown in parallel in hydrolysed casein medium at 37°C to an OD600 of 0.6. Sporulation was induced by resuspension in Sterlini-Mandelstam medium at 37C. Six hour and thirty minutes after resuspension, 25 ml of each culture were collected for RNA isolation and immediately mixed with an equal volume of methanol at –20C.
RNA was isolated with a Hot Phenol/Trizol procedure.
cDNA was prepared from 50 ug tot RNA and fluorescently labeled using Superscript Reverse Transcriptase and Cy5-dUTP for the RNA sample from strain PY79 and Cy3-dUTP for the RNA sample from strain PE454.
Protocols for RNA isolation, cDNA preparation and labeling, hybridization to the microarrays and washing procedure can be found at http://mcb.harvard.edu/losick
This is the third repetition of the experiment at this time point.


title: IFN 8c Chip B


title: IFN 6c Chip B


title: Control 1a Chip B


title: MelR slide 3

description: RNA was isolated from WAM132 melR cells, carrying plasmid pJW15 encoding mutant MelR FY53 KR182 (MelR*) and a control ‘no melR’ plasmid p121U9 (MelR -), grown in the absence of melibiose in M63 minimal media supplemented with fructose and amino acids (Webster et al., 1987 Gene 59: 253-263) to OD 0.5.

source_name: melR*

source_name: melR -


title: Control 4a Chip B


title: IFN 6a Chip B


title: Control 2b Chip B


title: MelR slide 2


title: Control 4c Chip B


title: Control 1b Chip B


title: Control 2a Chip B


title: Control 4b Chip B


title: IFN 5b Chip B


title: Control 2c Chip B


title: Control 1c Chip B


title: MelR slide 1


title: e1370df50 Sample 1

description:
Sample is also known as E1370mgdf50 1
Genotype is glp-4(bn2) daf-16(mgdf50); daf-2(e1370)
Nematodes were prepared for RNA extraction as follows. Eggs from each strain were first isolated from three large (9 cm) plates of just-starved, mixed-stage populations by alkaline hypochlorite treatment. These were allowed to hatch overnight in 50 ml S-media in Erlenmeyer flasks, at 20ºC with rotary shaking (200 rpm). The resulting synchronized L1 larvae were concentrated by centrifugation and pipetted onto eight large plates (one biological replicate), and incubated at 15º C. When most animals had reached L4 stage, plates were shifted up to 25ºC. Worms were harvested for RNA extraction the following day (1 day old, sterile adults), by washing worms off plates with M9, followed by three additional washes with ice-cold M9 to remove residual bacteria.  Total RNA was isolated using Trizol reagent, followed by purification and concentration using Qiagen RNEasy columns according to manufacturers' instructions (Qiagen, www1.qiagen.com). The quality of RNA samples was confirmed on an Agilent Bioanalyser 2100 (Agilent technologies, USA; www.chem.agilent.com).  All Affymetrix protocols were performed at the UCL Institute of Child Health Gene Microarray Centre. cRNA probe generated using standard Affymetrix protocols (www.affymetrix.com). Fragmented biotinylated probe was then hybridised to C. elegans whole genome arrays. Washing, labelling (streptavidin-phycoerythrin) and scanning followed standard procedures at the ICH Gene Microarray Centre.  Raw image files were converted to probe set data (.cel files) in Microarray Suite (MAS 5.0). The 20 probe set data files (5 replicates of four genotypes) were normalized together, and expression values were determined, using the Robust Multi-chip Average (RMA) method, implemented in the Affymetrix package (version 1.4.14) of the free statistical programming language R (www.r-project.org).
Keywords = Caenorhabditis, elegans, aging

source_name: Whole animal


title: IFN 8c Chip A


title: Control 4c Chip A


title: IFN 8a Chip A


title: IFN 6b Chip A


title: Control 4b Chip A


title: Control 1c Chip A