title: Testis_control untreated_rep1

growth_protocol: male mice were acclimatized and caged in groups of six or less at our in house facility. Animals were sacrificed by cervical dislocation, testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until RNA  extraction.

tag: 10 week old hpg males were killed  by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 1


title: Testis_control untreated_rep7

status: Public on Aug 18 2009

type: RNA

city: Oxford

first: Dilair

last: Baban

address: Roosevelt Drive

department: Wellcome Trust Centre Human Genetics

country: United Kingdom

institute: University of Oxford

description: Here we have addressed important question of changes in the global gene expression profile of testicular tissue following hormone stimulation (rFSH) for 12, 24 and 72h following injections compared to control untreated hpg males of the same age.

hyb_protocol: Fragmented labelled cRNA (15µg) was used in 300µl hybridization cocktail containing spiked controls (Affymetrix #900299). An aliquot of 200µl of this hybridization cocktail was used on each chip which was incubated at 45°C for 16 h in the hybridization oven, rotating at 60 rpm. Following hybridization, the arrays were processed using a Genechip Fluidics Station 400 according to recommended protocols (EukGE-WS2v4, Affymetrix) of double-staining and post-hybridisations washes.

scan_protocol: Fluorescent images were captured using gene Array Scanner 2500 and GCOS1.2 software (Affymetrix)

data_processing: We have used the GeneChip Robust Multi-array Average expression measurements (GC-RMA) as implemented in BioConductor R statistics (www.bioconductor.org). Cell Intensity files (CEL) were used to obtain expression values for all the 22,690 probe sets (transcripts) on each of these chips

label: Biotin

molecule: total RNA

organism: Mus musculus

source_name: Testis

label_protocol: 8 µg of RNA was converted to double-stranded cDNA using one-cycle cDNA synthesis kit (Affymetrix), followed by in vitro transcription (IVT) to generate biotin-labelled cRNA probes.

growth_protocol: male mice were acclimatized and caged in groups of six or less at our in house facility. Animals were sacrificed by cervical dislocation, testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until RNA extraction.

extract_protocol: Total RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions.

treatment_protocol: Control untreated

tag: 10 week old hpg males were killed by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 7


title: Testis_control untreated_rep4

tag: 10 week old hpg males were killed by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 4


title: Testis_control untreated_rep6

tag: 10 week old hpg males were killed by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 6


title: EFTF injected stage 15 Xenopus Laevis animal cap 1

status: Public on Aug 19 2009

city: Syracuse

first: Michael

middle: E

last: Zuber

state: NY

address: 766 Irving Ave

department: Ophthalmology

country: USA

institute: SUNY Upstate Medical University

description:
Carried out at SUNY-MAC (Syracuse, NY) per manufacturers
protocol (Affymetrix).

data_processing:
GeneSpring GX, ver. 7.3.1 (Agilent Technologies) was used for analysis. Cel file
data was pre-processed using RMA method with the average signal intensity normalized to primitive ectoderm isolated from Venus YFP RNA injected embryos in the case of EFTF-injected caps.

label: Biotin Labelling

molecule: polyA RNA

organism: Xenopus laevis

source_name: EFTF injected animal cap


title: Testis_control untreated_rep3

tag: 10 week old hpg males were killed by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 3


title: Testis_rFSH injected_72h_rep2

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 72h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 2


title: Testis_control untreated_rep2

tag: 10 week old hpg males were killed by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 2


title: Testis_rFSH injected_72h_rep1

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 72h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 1


title: Testis_rFSH injected_24h_rep2

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 24h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 1.


title: Testis_rFSH injected_72h_rep4

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 72h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 4


title: Testis_rFSH injected_24h_rep1

treatment_protocol: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 24h.

tag: 0 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 24h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 1.


title: Testis_rFSH injected_24h_rep3

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 24h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 3


title: Testis_rFSH injected_72h_rep3

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 72h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 3


title: Testis_rFSH injected_12h_rep4

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 12h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 4.


title: Testis_rFSH injected_12h_rep3

source_name: testis

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 12h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 3.


title: Testis_rFSH injected_12h_rep2

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 12h. Mice were killed one hour after the last injection by cervical dislocation; testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 2.


title: Wakulla-infected SCID mouse liver_Sample153

status: Public on Aug 17 2009

city: Columbus

first: Koshiro

last: Miura

state: OH

address: 1900 Coffey Rd

department: Veterinary Biosciences

institute: The Ohio State University

description: 3 mice were used for this experiment.

hyb_protocol: The labeled cRNA was hybridized to GeneChip Mouse Genome 430 2.0 Array (Affymetrix) containing over 39,000 element-spotted murine cDNA microarrays.

scan_protocol: After washing and staining with streptoavidin phycoerythrin, the array was scanned with GeneChip Scanner 3000 7G (Affymetrix).

data_processing: Data analysis was performed with Partek Discovery Suite (Partek Inc., St. Louis, MO). The average differences obtained with probes derived from liver samples infected with each of three strains of E. chaffeensis, were base-lined against average differences obtained from sham-infected liver samples to generate the -fold difference value for each gene call.

label: phycoerythrin

source_name: Wakulla-infected SCID mouse liver 15 dpi

label_protocol: About 8 ug of each RNA sample was applied to double-strand cDNA preparation using GeneChip One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA). Biotin-labeled cRNA was generated from the cDNA using GeneChip IVT Labeling Kit (Affymetrix).

extract_protocol: Total RNAs were extracted from E. chaffeensis-infected or uninfected mouse liver using RNeasy Mini Kit (QIAGEN).

tag: SCID mouse, male, 7 weeks, 15 days after inoculation of E. chaffeensis Wakulla


title: Liberty-infected SCID mouse liver_Sample118

description: 3 mice were used for this experiment.

source_name: Liberty-infected SCID mouse liver 15 dpi

tag: SCID mouse, 7 weeks, male, 15 days after inoculation


title: Arkansas-infected SCID mouse liver_Sample115

description: 3 mice were used for this experiment.

source_name: Arkansas-infected SCID mouse liver 15 dpi

tag: SCID mouse, male, 7 weeks, 15 days after inoculation


title: Liberty-infected SCID mouse liver_Sample119

description: 3 mice were used for this experiment.


title: Wakulla-infected SCID mouse liver_Sample151

description: 3 mice were used for this experiment.


title: Wakulla-infected SCID mouse liver_Sample152

description: 3 mice were used for this experiment.


title: Liberty-infected SCID mouse liver_Sample121

description: 3 mice were used for this experiment.


title: Mock-infected SCID mouse liver_Sample141

description: 3 mice were used for this experiment.

source_name: Mock-infected SCID mouse liver; control.

tag: SCID mouse, male, 7 weeks


title: Lateral endoderm 3

hyb_protocol: Carried out at SUNY-MAC (Syracuse, NY) per manufacturers protocol (Affymetrix).

data_processing:
GeneSpring GX, ver. 7.3.1 (Agilent Technologies) was used for analysis. Cel file
data was pre-processed using RMA method with the average signal intensity normalized to stage 15 whole embryos when analyzing EP, PNP and LE samples.

source_name: Stage 15 Xenopus Laevis lateral endoderm


title: Testis_rFSH injected_12h_rep1

tag: 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 12h. Mice were killed one hour after the last injection by cervical dislocation, testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the the manufacturer’s instructions manufacturer’s instructions. Biological Replicate 1.


title: Arkansas-infected SCID mouse liver_Sample113

description: 3 mice were used for this experiment.


title: Arkansas-infected SCID mouse liver_Sample114

description: 3 mice were used for this experiment.


title: Mock-infected SCID mouse liver_Sample142

description: 3 mice were used for this experiment.


title: Mock-infected SCID mouse liver_Sample143

description: 3 mice were used for this experiment.


title: Patient 32948 (HsO2F-041_016315)

status: Public on Aug 05 2009

city: Luxembourg

first: Yvan

last: Devaux

address: 120 route d'Arlon

country: Luxembourg

institute: LIH

description:
cy3 vs cy5
Date:2006/11/27
plasma procalcitonin (PCT) level: 0.26

hyb_protocol: Agilent : 750.0 ng at 60 degree_C during 17 hours

scan_protocol: Scanned on an GenePix 4000B fluorescent scanner. Image intensity data were extracted with GenePix Pro 6.0 analysis software.

data_processing: Log (base 2) transform of the ratio of the medians.

organism: Homo sapiens

label_protocol: Ambion

extract_protocol: Qiagen

value: PAXgene

tag: Tissue

value: Blood

extract_protocol: Trizol

value: Trizol

value: Ref

value: Tous


title: Patient 33104 (HsO2E-107_016291)

description:
Cy3 vs cy5
Date:2006/11/20
plasma procalcitonin (PCT) level: 0.12

tag: Origin

value: human


title: Patient 34374 (HsO2F-043_016421)

description:
cy3 vs cy5
Date:2006/11/28
plasma procalcitonin (PCT) level: 0.39


title: Patient 33650 (HsO2F-036_016404)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 22.5


title: Patient 33650 (HsO2F-037_016407)


title: Patient 33079 (HsO2E-105_016294)

description:
cy3 vs cy5
Date:2006/11/21
plasma procalcitonin (PCT) level: 30.82


title: Patient 32794 (HsO2E-074_016250)

description:
Cy3 vs Cy5
Date:2006/11/07
plasma procalcitonin (PCT) level: 3.21


title: Patient 32948 (HsO2F-042_016316)

description:
cy3 vs Cy5
Date:2006/11/27
plasma procalcitonin (PCT) level: 0.26


title: Patient 32948 (HsO2F-040_016313)


title: Patient 33406 (HsO2F-038_016311)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 0.18


title: Patient 34076 (HsO2F-076_016276)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 0.23


title: Patient 31715 (HsO2E-072_016207)

description:
Cy3 vs Cy5
Date:2006/11/07
plasma procalcitonin (PCT) level: 7.96


title: Patient 31715 (HsO2E-073_016193)

description:
Cy3 vs Cy5
Date:2006/11/09
plasma procalcitonin (PCT) level: 7.96


title: Patient 32948 (HsO2F-039_016312)


title: Patient 32794 (HsO2E-075_016247)


title: Patient 34076 (HsO2F-077_016277)


title: Patient 34076 (HsO2F-079_016223)


title: Patient 32380 (HsO2E-011_016170)

description:
Cy3 vs Cy5
Date:2006/10/26
plasma procalcitonin (PCT) level: 0.32


title: Patient 32794 (HsO2E-076_016246)


title: Patient 32380 (HsO2E-013_016112)

description:
Cy3 vs Cy5
Date:2006/11/10
plasma procalcitonin (PCT) level: 0.32


title: Patient 34076 (HsO2F-078_017265)


title: Patient 30516 (HsO2C-021_013185)

description:
Cy3 vs cy5
Date:2006/08/30
plasma procalcitonin (PCT) level: 47

value: Réf


title: Patient 32047 (HsO2E-078_016361)

description:
Cy3 vs cy5
Date:2006/10/12
plasma procalcitonin (PCT) level: 2.88


title: Patient 32047 (HsO2E-080_016300)

description:
cy3 vs Cy5
Date:2006/10/12
plasma procalcitonin (PCT) level: 2.88


title: Patient 32047 (HsO2E-077_016245)


title: Patient 33104 (HsO2E-106_016292)


title: Patient 31456 (HsO2E-079_016297)

description:
cy3 vs cy5
Date:2006/10/16
plasma procalcitonin (PCT) level: 0.34


title: Patient 32794 (HsO2E-014_016438)


title: Patient 33676 (HsO2F-080_016221)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 0.34


title: Patient 32380 (HsO2E-012_016111)


title: Patient 33406 (HsO2F-049_016175)


title: Patient 33406 (HsO2F-050_016176)


title: Patient 30516 (HsO2C-022_013087)

description:
Cy3 vs Cy5
Date:2006/08/30
plasma procalcitonin (PCT) level: 47


title: Patient 33406 (HsO2F-051_016177)


title: Patient 32997 (HsO2F-053_017218)

description:
cy3 vs cy5
Date:2006/11/30
plasma procalcitonin (PCT) level: 0.21


title: Patient 32997 (HsO2F-052_017213)


title: Patient 32233 (HsO2E-085_016401)

description:
Cy3 vs Cy5
Date:2006/10/19
plasma procalcitonin (PCT) level: 0.19


title: Patient 32997 (HsO2F-055_016368)

description:
Cy3 vs Cy5
Date:2006/11/30
plasma procalcitonin (PCT) level: 0.21


title: Patient 30516 (HsO2C-023_012843)


title: Patient 31398 (HsO2E-087_016379)

description:
Cy3 vs cy5
Date:2006/10/17
plasma procalcitonin (PCT) level: 0.27


title: Patient 30393 (HsO2C-027_012850)

description:
cy3 vs cy5
Date:2006/08/28
plasma procalcitonin (PCT) level: 70.6


title: Patient 32047 (HsO2E-082_016340)


title: Patient 31398 (HsO2E-086_016380)


title: Patient 32233 (HsO2E-088_016378)


title: Patient 32262 (HsO2E-089_016295)

description:
Cy3 vs Cy5
Date:2006/10/24
plasma procalcitonin (PCT) level: 4.24


title: Patient 30516 (HsO2C-024_012845)


title: Patient 30393 (HsO2C-029_013129)


title: Patient 32262 (HsO2E-091_016322)


title: Patient 32233 (HsO2E-084_016402)


title: Patient 33676 (HsO2F-065_016370)


title: Patient 32997 (HsO2F-056_016366)


title: Patient 30341 (HsO2C-025_012846)

description:
cy3 vs cy5
Date:2006/08/23
plasma procalcitonin (PCT) level: 54.1


title: Patient 30393 (HsO2C-028_013128)


title: Patient 32262 (HsO2E-090_016296)

description:
Cy3 vs Cy5
Date:2006/11/10
plasma procalcitonin (PCT) level: 4.24


title: Patient 34374 (HsO2F-057_016365)

description:
cy3 vs cy5
Date:2006/11/28
plasma procalcitonin (PCT) level: 0.39


title: Patient 34374 (HsO2F-058_017170)


title: Patient 34374 (HsO2F-059_017194)


title: Patient 32262 (HsO2E-092_016323)


title: Patient 30393 (HsO2C-026_012847)


title: Patient 33079 (HsO2E-093_016326)

description:
cy3 vs cy5
Date:2006/11/13
plasma procalcitonin (PCT) level: 30.82


title: Patient 31715 (HsO2E-097_016273)

description:
Cy3 vs Cy5
Date:2006/11/09
plasma procalcitonin (PCT) level: 7.96


title: Patient 33079 (HsO2E-095_016279)

description:
Cy3 vs cy5
Date:2006/11/13
plasma procalcitonin (PCT) level: 30.82


title: Patient 31398 (HsO2E-098_016274)

description:
cy3 vs cy5
Date:2006/10/26
plasma procalcitonin (PCT) level: 0.27


title: Patient 31715 (HsO2E-096_016272)


title: Patient 33676 (HsO2F-067_016349)


title: Patient 33854 (HsO2F-068_016341)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 1.4

value: blood


title: Patient 33854 (HsO2F-070_016413)


title: Patient 31398 (HsO2E-099_016139)

description:
cy3 vs cy5
Date:2006/10/17
plasma procalcitonin (PCT) level: 0.27


title: Patient 33079 (HsO2E-094_016280)


title: Patient 31456 (HsO2E-100_016396)

description:
cy3 vs cy5
Date:2006/10/16
plasma procalcitonin (PCT) level: 0.34


title: Patient 33854 (HsO2F-069_016412)


title: Patient 30070 (HsO2C-098_012636)

description:
cy3 vs cy5
Date:2006/08/21
plasma procalcitonin (PCT) level: 11.4


title: Patient 30341 (HsO2C-099_012781)

description:
cy5 vs cy5
Date:2006/08/23
plasma procalcitonin (PCT) level: 54.1


title: Patient 30070 (HsO2C-103_012468)


title: Patient 33650 (HsO2F-035_016406)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 22.5


title: Patient 31456 (HsO2E-101_016395)


title: Patient 32380 (HsO2E-010_016167)

description:
Cy3 vs Cy5
Date:2006/11/10
plasma procalcitonin (PCT) level: 0.32


title: Patient 33676 (HsO2F-066_016342)

description:
cy3 vs cy5
Date:2006/12/20
plasma procalcitonin (PCT) level: 0.34


title: Patient 31456 (HsO2E-102_016392)


title: Patient 33854 (HsO2F-071_016190)


title: Patient 30341 (HsO2C-101_012408)


title: Patient 30070 (HsO2C-102_012467)


title: Patient 30070 (HsO2C-097_012606)


title: Patient 30341 (HsO2C-100_012477)


title: Patient 33650 (HsO2F-034_016405)


title: Patient 33104 (HsO2E-103_016393)

description:
Cy3 vs cy5
Date:2006/11/20
plasma procalcitonin (PCT) level: 0.12


title: Patient 33104 (HsO2E-104_016394)


title: Baker's yeast_freeze_7day_rep2

status: Public on Jul 31 2009

city: Tsukuba

first: Satomi

middle: Mizukami

last: Murata

address: Kannondai 2-1-12

country: Japan

institute: National Food Research Institute

description: We loaded freeze injuries into baker's yeast cells under different freezing period and analyzed gene expressions during recovery process from freeze injured stated.

hyb_protocol: Affymetrix Yeast Genome S98 arrays were used as a DNA microarray.  The probe was hybridized to a DNA microarray at 45oC for 18 h according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA).

scan_protocol: The washing and staining of arrays were performed using a GeneChip Fluidics Station 400.  The scanning of arrays was carried out using a GeneChip scanner (Affymetrix).

data_processing: The fluorescence intensities of array images were quantified using the Affymetrix software package (GeneChip Operating Software 1.2).  Statistical analysis after data acquisition and normalization of expression data were carried out using GeneSpring ver.7 (Agilent technologies, Palo Alto, USA).  After data transformation (set measurements less than 0.01 to 0.01) to GeneSpring, per chip normalization to the 50th percentile was performed.  Per gene normalization to control samples (freezing time was 0day) was applied to the per chip normalized values.

organism: Saccharomyces cerevisiae

source_name: Baker’s yeast, freeze, 7day

label_protocol: cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA).

extract_protocol: RNA was extracted from the yeast cells using the hot phenol method.  Poly(A)+  RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan).

treatment_protocol: The compressed yeast was suspended in liquid fermentation (LS) without sucrose and maltose LF-S medium (10 ml) at the concentration of OD600=30, and the suspension was frozen at -20oC for 7 day.  The frozen samples were thawed at 30oC for 10 min in a water bath.

tag: Commercial products of compressed yeast for bread making


title: Baker's yeast_freeze_3day_rep2

source_name: Baker’s yeast, freeze, 3day

treatment_protocol: The compressed yeast was suspended in liquid fermentation (LS) without sucrose and maltose LF-S medium (10 ml) at the concentration of OD600=30, and the suspension was frozen at -20oC for 3 day.  The frozen samples were thawed at 30oC for 10 min in a water bath.


title: Baker's yeast_freeze_0day_rep1

source_name: Baker’s yeast, freeze, 0day

treatment_protocol: The compressed yeast was suspended in liquid fermentation (LS) without sucrose and maltose LF-S medium (10 ml) at the concentration of OD600=30, and the suspension was frozen at -20oC for 0 day.  The frozen samples were thawed at 30oC for 10 min in a water bath.


title: Baker's yeast_freeze_7day_rep1


title: Baker's yeast_freeze_0day_rep2


title: Patient 32233 (HsO2E-083_016339)

description:
Cy3 vs Cy5
Date:2006/10/19
plasma procalcitonin (PCT) level: 0.19


title: Baker's yeast_freeze_14day_rep2

source_name: Baker’s yeast, freeze, 14day

treatment_protocol: The compressed yeast was suspended in liquid fermentation (LS) without sucrose and maltose LF-S medium (10 ml) at the concentration of OD600=30, and the suspension was frozen at -20oC for 14 day.  The frozen samples were thawed at 30oC for 10 min in a water bath.


title: Carrier biological rep3 (Triazolopyrimidine)

status: Public on Jul 22 2009

city: Corvallis

first: Malay

last: Das

state: OR

address: 200 SW 35th STreet

department: EEB

institute: National Research Council, US-EPA

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip ATH1 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using an Affymetrix GeneChip scanner 3000 at 570 nm.

data_processing: Intensities of the fluorescence emission were captured and array image was visually screened for signal artifacts

label: biotin

organism: Arabidopsis thaliana

source_name: Leaf, surfactant (preference, 0.25%) 24h post tretment

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 4 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Seeds of A. thaliana, col 0 were stratified at 3-4C for 2-4 days and then grown under 22-23C, 16h photoperiod and 175-210 micromol per sec light intensity

extract_protocol: Triazol extraction of total RNA and subsequent purification with Rneasy mini spin columns

treatment_protocol: 14 days old plants were sprayed with surfectant and herbicide solutions in a RIC spray chamber (RC-5000-100EP)

tag: No visible symptoms of injury observed


title: Carrier biological rep2 (Imidazolinone)


title: Carrier biological rep1 (Triazolopyrimidine)


title: Carrier biological rep2 (Triazolopyrimidine)


title: Carrier biological rep1 (Imidazolinone)


title: Carrier biological rep3 (Imidazolinone)


title: Baker's yeast_freeze_14day_rep1


title: Hela_Ctrl3

status: Public on Jul 17 2009

city: Bures-sur-Yvette

first: Arndt

last: Benecke

address: 35 route des Chartres

department: Integrative Biology

country: France

institute: Institut des Hautes Etudes Scientifiques & I.R.I.-CNRS

description: The Bcl-xS transcription program.

hyb_protocol: Array hybridization was performed for 16 hrs at 55 oC. Pre-Hybridization, Hybridization, antibody binding and Chemiluminescence reaction were performed using Applied Biosystems Chemiluminescence Detection Kit (P/N 4342142) following the manufacturer’s protocol (P/N 4339627).

scan_protocol: Image acquisition and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit (P/N 4342142) and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer 1.1.1 (P/N 4338036) following the manufacturer’s protocol (P/N 4339629).

data_processing: Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software v1.1.1 (P/N 4336391). Median normalization and Probabilty for a gene to be expressed(A) were calculated with proprietary software.

label: DIG-UTP

source_name: Hela cell line

label_protocol: Digoxigenin-UTP labeled cRNA was generated and amplified from 2 μg of total RNA from each sample using Applied Biosystems NanoAmp RT-IVT Labeling Kit (P/N 4365715) according to the manufacturer’s protocol(P/N 4365710).

extract_protocol: RNA was extracted from the cell lines using RNeasy Midi Kit (Quiagen) according to the instructions provided by the manufacturer

tag: Hela cells + Control antisense RNA oligo


title: Diabetes-exposed embryo with neural tube defect 2

status: Public on Jul 21 2009

city: Baton Rouge

first: Claudia

last: Kappen

state: LA

address: 6400 Perkins Road

department: Developmental Biology

institute: Pennington Biomedical Research Center

description: Gene expression data from embryos exposed to maternal diabetes and analyzed at embryonic day 10.5

hyb_protocol: Probes were hybridized to the Affymetrix  430A 2.0 genome chip (Santa Clara, CA). Following washing and staining per established manufacturer protocols for Affymetrrix arrays.

scan_protocol: The chips were scanned using the Affymetrix GeneChip 3000 scanner in the UNMC microarray core facility.  Images were analyzed using Affymetrix GCOS imaging software.  Quality metric parameters including noise level, background, and the efficiency of reverse transcription were ascertained for all hybridizations. Data sets passing the stringent quality recommendations were further analyzed to identify differentially expressed genes.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

source_name: embryo, E10.5, diabetes-exposed

label_protocol: Five ug  of total RNA was reverse-transcribed, and biotin-labeled cRNA probe generated using the Affymetrix labeling kit and protocol per manufacturers instructions.

extract_protocol: Total RNA was isolated from whole embryos at embryonic day 10.5 (E10.5) using Trizol® (Invitrogen, Carlsbad, CA).

treatment_protocol: Embryos of the appropriate stage E10.5 were dissected under the dissecting scope. Selected embryos were rinsed with PBS  and placed on ice in the Trizol solution (Invitrogen).

tag: Strain

value: FVB

tag: Age

value: embryonic day 10.5

value: whole embryo

tag: disease exposure

value: diabetes

tag: congenital malformations

value: neural tube defects (NTD)


title: Diabetes-exposed embryo with no neural tube defect 1

value: none identified


title: Hela-Bclx2

description: The Bcl-xS transcription program.

tag: Hela cells + Bclx-exon 2 antisense RNA oligo


title: Control embryo 2

description: Gene expression data from embryos from normal pregnancy analyzed at embryonic day 10.5

source_name: embryo, E10.5, normal pregnancy

tag: normal

tag: noram


title: Diabetes-exposed embryo with neural tube defect 1


title: Control embryo 1


title: Hela-Bclx3

description: The Bcl-xS transcription program.


title: Hela-Bclx1

description: The Bcl-xS transcription program.


title: Diabetes-exposed embryo with no neural tube defect 3


title: Hela_Ctrl2

description: The Bcl-xS transcription program.


title: Diabetes-exposed embryo with no neural tube defect 2


title: Baker's yeast_freeze_3day_rep1


title: Hela_Ctrl1

description: The Bcl-xS transcription program.

scan_protocol: image acquisition and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit (P/N 4342142) and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer (P/N 4338036) following the manufacturer’s protocol (P/N 4339629).

data_processing: Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software v 1.1 (P/N 4336391). Median normalization and Probabilty for a gene to be expressed(A) were calculated with proprietary software.

label_protocol: Digoxigenin-UTP labeled cRNA was generated and amplified from 2 μg of total RNA from each sample using Applied Biosystems NanoAmp RT-IVT Labeling Kit (P/N 4365715)  according to the manufacturer’s protocol(P/N 4365710).


title: Cu_600uM_12h_Rep 4

status: Public on Jun 25 2009

city: RTP

first: Min Ok

last: Song

state: NC

address: 111 T.W. Alexander Dr

department: DIR ETP

institute: NIEHS

description: Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 100ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer's protocol.    For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol.  Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol.    Chips were scanned with an Agilent Scanner and processed with the Agilent Feature Extraction Software.

hyb_protocol: The quantity and purity of the resulting fluorescently-labeled cRNA was evaluated using a Nanodrop ND-100 spectrophotometer (Nanodrop Technologies), and the size distribution using an Agilent Bioanalyzer.  Equal amounts of Cy3- and Cy5-lableled cRNA were then hybridized to an Agilent Human Microarray (~22,000 k features) for 17 h at 65°C.

scan_protocol: The hybridized microarrays were washed and scanned using an Agilent G2565BA scanner.  Data were extracted from the scanned images using Agilent Feature Extraction software.

data_processing: GeneSpring GX 7.3 (Agilent Technologies) was used to identify genes that showed significant changes in gene expression with any treatment.  For global normalization of raw microarray data, per spot and per chip: intensity dependent (Lowess) normalization was applied.

source_name: Control 12h

label_protocol: Total RNA (100 ng) from control or metal-treated cells was amplified and labeled with Cy3 or Cy5 fluorescent dye using Agilent Technologies Low RNA Input Linear Amplification labeling kit following the manufacture's protocol.

extract_protocol: Total RNAs were isolated from untreated and treated cells using RNeasy mini kits (Qiagen, Inc.) following manufacturer's instructions.  The quality of the purified RNA was determined using a BioAnalyzer (Agilent Technologies, Palo Alto, CA).

treatment_protocol: HepG2 cells were plated and allowed to grow until they were 50 % confluent.  Cells were then treated with 100, 200, 400 and 600 mM of copper sulfate for 4, 8, 12 and 24 h.

tag: Control

tag: Treated


title: Cu_600uM_12h_Rep 1


title: Cu_600uM_8h_Rep 2

source_name: Control 8h


title: Cu_600uM_8h_Rep 3


title: Cu_600uM_12h_Rep 3


title: Cu_600uM_24h_Rep 5

source_name: Control 24h


title: Cu_600uM_24h_Rep 6


title: Cu_600uM_24h_Rep 2


title: Cu_600uM_24h_Rep 1


title: Cu_600uM_12h_Rep 5


title: Cu_600uM_12h_Rep 2


title: Cu_600uM_24h_Rep 3


title: Cu_600uM_24h_Rep 4


title: Cu_600uM_12h_Rep 6


title: Cu_600uM_8h_Rep 1


title: Cu_400uM_24h_Rep 2


title: Cu_600uM_8h_Rep 6


title: Cu_600uM_4h_Rep 1

source_name: Control 4h


title: Cu_400uM_24h_Rep 1


title: Cu_600uM_4h_Rep 3


title: Cu_600uM_4h_Rep 6


title: Cu_600uM_8h_Rep 5


title: Cu_600uM_4h_Rep 2


title: Cu_600uM_4h_Rep 5


title: Cu_600uM_8h_Rep 4


title: Cu_600uM_4h_Rep 4


title: Cu_400uM_24h_Rep 4


title: Cu_400uM_24h_Rep 6


title: Cu_400uM_24h_Rep 5


title: Cu_400uM_24h_Rep 3


title: Cu_400uM_12h_Rep 1


title: Cu_400uM_12h_Rep 2


title: Cu_400uM_12h_Rep 4


title: Cu_400uM_12h_Rep 6


title: Cu_400uM_8h_Rep 1


title: Cu_400uM_8h_Rep 4


title: Cu_400uM_8h_Rep 3


title: Cu_400uM_12h_Rep 3


title: Cu_200uM_24h_Rep 2


title: Cu_400uM_8h_Rep 6


title: Cu_200uM_12h_Rep 2


title: Cu_400uM_4h_Rep 5


title: Cu_400uM_4h_Rep 1


title: Cu_200uM_24h_Rep 4


title: Cu_400uM_8h_Rep 5


title: Cu_400uM_12h_Rep 5


title: Cu_400uM_4h_Rep 7


title: Cu_200uM_24h_Rep 3


title: Cu_400uM_4h_Rep 6


title: Cu_400uM_4h_Rep 3


title: Cu_200uM_12h_Rep 1


title: Cu_400uM_8h_Rep 2


title: Cu_200uM_24h_Rep 1


title: Cu_200uM_24h_Rep 6


title: Cu_200uM_24h_Rep 5


title: Cu_400uM_4h_Rep 2


title: Cu_400uM_4h_Rep 4


title: Cu_400uM_4h_Rep 8


title: Cu_200uM_12h_Rep 3


title: Cu_200uM_12h_Rep 6


title: Cu_200uM_12h_Rep 4


title: Cu_200uM_12h_Rep 5


title: Cu_200uM_8h_Rep 1


title: Cu_200uM_8h_Rep 2


title: Cu_200uM_8h_Rep 3


title: Cu_200uM_8h_Rep 5


title: Cu_200uM_8h_Rep 6


title: Cu_200uM_4h_Rep 6


title: Cu_100uM_24h_Rep 6


title: Cu_100uM_24h_Rep 3


title: Cu_200uM_8h_Rep 4


title: Cu_100uM_24h_Rep 2


title: Cu_200uM_4h_Rep 5


title: Cu_200uM_4h_Rep 1


title: Cu_200uM_4h_Rep 4


title: Cu_100uM_12h_Rep 3


title: Cu_100uM_12h_Rep 1


title: Cu_100uM_24h_Rep 5


title: Cu_200uM_4h_Rep 3


title: Cu_100uM_24h_Rep 1


title: Cu_100uM_24h_Rep 4


title: Cu_100uM_12h_Rep 2


title: Cu_100uM_12h_Rep 6


title: Cu_100uM_12h_Rep 4


title: Cu_100uM_12h_Rep 5


title: Cu_200uM_4h_Rep 2


title: Cu_100uM_8h_Rep 2


title: Cu_100uM_8h_Rep 1


title: Cu_100uM_8h_Rep 4


title: Cu_100uM_8h_Rep 5


title: Cu_100uM_8h_Rep 6


title: Cu_100uM_8h_Rep 3


title: Cu_100uM_4h_Rep 2


title: Cu_100uM_4h_Rep 4


title: PAO1 cyclamate_sulfate 1

status: Public on Jun 22 2009

city: Manchester

middle: A.

last: Kertesz

address: Oxford Rd

department: Faculty of Life Sciences

institute: University of Manchester

description: no treatment

hyb_protocol: Standard Affymetrix procedures

scan_protocol: Standard Affymetrix procedures

data_processing: Analysis based on GC-RMA normalization of the data in the CEL files

organism: Pseudomonas aeruginosa

source_name: Pseudomonas aeruginosa PAO1 grown with cyclamate and sulfate as S source

label_protocol: Standard Affymetrix procedures, starting with 5-10 ug RNA

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. They wer then grown for at least 12 generations with 500 uM sulfate and 500 uM cyclamate (sodium cyclohexylsulfamate) as S source. These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM cyclamate/500 uM sulfate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.

extract_protocol: Qiagen RNeasy mini kit – bacterial protocol. The washing step with 700 ul of buffer RW1 was split up into 2 steps and an on column digest of DNA using Qiagen DNase was performed in between

treatment_protocol: no treatment

tag: wild type, sequenced strain


title: PAO1 sulfate 1

description: no treatment

source_name: Pseudomonas aeruginosa PAO1 grown with sulfate as S source

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. .These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM sulfate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: E601 mucin 2

description: no treatment

source_name: Pseudomonas aeruginosa E601 grown with human colon cell line mucin LS174T as S source

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with 25 ug/ml human colon cell line mucin LS174T as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 25 ug/ml LS174T mucin as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.

tag: Pseudomonas aeruginosa strain E601, a cystic fibrosis isolate with mucin sulfatase activity


title: E601 mucin_cyclamate 3

description: no treatment

source_name: Pseudomonas aeruginosa E601 grown with cyclamate and human colon cell line mucin LS174T as S source

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with a combination of 500 uM cyclamate (sodium cyclohexylsulfamate) and 25 ug/ml human colon cell line mucin LS174T as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with the same mixture of cyclamate and mucin as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: Cu_100uM_4h_Rep 5


title: E601 mucin 3

description: no treatment


title: Cu_100uM_4h_Rep 1


title: E601 cyclamate 2

description: no treatment

source_name: Pseudomonas aeruginosa E601 grown with cyclamate as S source

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with 500 uM cyclamate (sodium cyclohexylsulfamate) as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM cyclamate as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: Cu_100uM_4h_Rep 3


title: PAO1 sulfate 2

description: no treatment


title: E601 cyclamate 3

description: no treatment


title: PAO1 sulfate 3

description: no treatment

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM sulfate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: E601 mucin_cyclamate 2

description: no treatment


title: PAO1 cyclamate_sulfate 2

description: no treatment

source_name: Pseudomonas aeruginosa PAO1 grown with sulfate and cyclamate as S source


title: Cu_100uM_4h_Rep 6


title: E601 mucin_cyclamate 1

description: no treatment

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. The cells were then grown for at least 12 generations with a combination of 500 uM cyclamate (sodium cyclohexylsulfamate) and 25 ug/ml human colon cell line mucin LS174T as sulfur source.These cells were then used to inoculate the starter culture needed for RNA preparation, grown with the same mixtire of cyclamate and mucin as S source). For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: E601 mucin 1

description: no treatment


title: E601 cyclamate 4

description: no treatment


title: E601 sulfate 3

description: no treatment

source_name: Pseudomonas aeruginosa E601 grown with sulfate as S source

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. These cells were then used to inoculate the starter culture needed for RNA preparation. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: PAO1 cyclamate 1

description: no treatment

source_name: Pseudomonas aeruginosa PAO1 grown with cyclamate as S source

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. They were then grown for at least 12 generations with 500 uM cyclamate (sodium cyclohexylsulfamate) as S source. These cells were then used to inoculate the starter culture needed for RNA preparation, grown with 500 uM cyclamate as S source. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2.


title: PAO1 cyclamate 2

description: no treatment


title: E601 cyclamate 1

description: no treatment


title: E601 sulfate 2

description: no treatment

extract_protocol: Qiagen RNeasy mini kit – bacterial protocol. The washing step with 700ul of buffer RW1 was split up into 2 steps and an on column digest of DNA using Qiagen DNase was performed in between


title: E601 sulfate 1

description: no treatment

source_name: Pseudomonas aeruginosa cells

growth_protocol: 5 ml cultures of cells were grown aerobically in succinate-based minimal medium in 50 ml flasks shaken with 220 rpm at 37 C. Cells were grown with 500 uM Sulfate in repeated batch cultures for at least 20 generations. These cells were then used to inoculate the starter culture needed for RNA preparation. For the preparation of RNA a 5 ml culture was started with 100 ul of a freshly grown culture. and grown to an OD600 of 0.2

tag: Pseudomonas aeruginosa strain E601 - cystic fibrosis oisolate with mucin sulfatase activity


title: Listeria monocytogenes LMB0622_acid adaptation_rep1

status: Public on Jun 15 2009

city: Pullman

first: Devendra

middle: H

last: Shah

state: WA

address: 100 Dairy Rd

department: Veterinary Microbiology and Pathology

institute: Washington State University

description:
Slide Bar Code: 00088018
There were three distinct stresses: acid adaptation (pH5.5); acid stress (pH 3.5); bile stress (0.3% Bile/pH 5.5).
All strains underwent 3 biological replicates.  Biological replicates that did not correlate with each other (pearson correlation >0.6) were discarded from downstream analysis.

hyb_protocol: The labeled cDNA pellets were resuspended in 70uL of hybridization buffer (5x denhardt’s/4x SSC) and boiled for 2 minutes.  Samples were centrifuged for 1 minute and applied to the slides under Erie Scientific Lifter slips (24x60mM).  Hybridizations were conducted in a 45oC water bath in corning hybridization chambers for 18 hours.  Post hybridization washes consisted of three 1 minute TNT (Tris/NaCl/Tween) washes (gently shaking).  The slides were dried using a Microarray High-Speed Centrifuge (TeleChem International, Inc).

scan_protocol: Applied Precisions ArrayWorx Scanner employing the following settings: CY3 for 0.1s and A488 for 0.3s with a resolution of 9.6um.

data_processing: TIFF images were processed using SPOTfinder software (TIGR TM4 software suite) using the otsu spot setting and employing background subtraction within the spotfinding software.  The resultant median intensities were imported into MATlab software (Mathworks, Inc) for lowess normalization.  The normalized intensities and ratios were imported into an access database.  Spot quality control filtering was conducted in access (Microsoft) and consisted of the following criterion:  1) Signal:Noise ratio must be greater than 2 (A488) or 10 (A555); 2) The mean spot intensity must be within 25% of the median spot intensity; 3) The coefficient of variance for replicate spots must be less than 0.2; 4)Those spots with only a single channel passing the previous three filters were then low-intensity filtered with a cutoff of 4000.

label: AlexaFluor 488

organism: Listeria monocytogenes

source_name: LMB0622, neutral pH

label_protocol:
cDNA was generated as follows: 3μg of total RNA and 150ng of random hexamers (RHs) were combined and heated to 70oC for 10 minutes.  This RNA:RHs mixture was snap cooled on ice for 1 minute.  The remaining reagents from the SuperScriptTM III First Strand Synthesis Kit® (Invitrogen) were added to the following final concentrations: 1.2x RT Buffer; 6.1mM MgCl2; 12mM DTT; 2.7mM dNTP’s (Promega) which contained aa-dUTP (Ambion) at a ratio of 3.5:1; 40U RNaseOUTTM; and 760U SuperScriptTM III.  This mixture was incubated for 18h at 42oC.  Following the 42oC incubation, EDTA (pH 8.0) and NaOH were added to a final concentration of 0.1M and 0.2M, respectively.  This mixture was incubated at 65oC for 15 minutes in order to hydrolyze the RNA.  The solution was neutralized by the addition of 1M Tris-HCl pH 7.0 to a final concentration of 0.3M.  The cDNA was cleaned up using a QiaQuick PCR clean-up column (Qiagen) with the following modifications:  all spins except the elution step were conducted at 6,000 rpms and the column drying spin (step 6) was extended to 5 minutes; the wash buffer and elution buffers consisted of 5mM KPO4/80% EtOH and 4mM KPO4, respectively, to avoid Tris contamination in the cDNA; finally, the elution buffer was preheated to 37oC and the column was incubated at 37oC for 5 minutes prior to elution from the column.  The eluted cDNA was dried down in a centrivap for 45 minutes at 40oC.  The resultant pellet was either stored at -20oC until later use or resuspended in 5ul of water and incubated for 2-3 hours at 55oC in preparation for a labeling reaction.
The labeling reactions were performed utilizing AlexaFluor 555 and AlexaFluor 488 as per manufacturer’s instructions with the minor modification of an overnight incubation (instead of 1 hour).    Labeling reactions were cleaned up as described above for cDNA clean-up, with the replacement of the KPO4 buffers with manufacturer supplied buffers.  The eluted labeled cDNA was quantified on a spectrophotometer.  cDNA concentrations were calculated and combined such that the same amount of cDNA was used for each condition (control vs. stress).  Each hybridization consisted of between  2.4 – 4.6nM cDNA (per condition) and 60 – 480 pmol dye incorporation (per dye)

growth_protocol: All strains were passaged 3 times in BHI at 37oC with moderate shaking for 18 hours prior to stress protocol.

extract_protocol: RNA was extracted using TRIzol (Invitrogen) as per the manufacturer’s instructions with only minor modifications.  Briefly, bacterial pellets were resuspended in 3-mL of TRIzol followed by the addition of 0.1mm Zirconia beads (BIOSPEC Products Inc) for cell disruption.  This mixture underwent rapid horizontal vortexing for 1 hour at 4oC.  The beads were pelleted out by centrifugation and chloroform was added for organic extraction.  Phase separation was achieved through centrifugation at which time the aqueous phase was removed to a separate tube.  The organic phase was back-extracted by the addition of ultra-pure RNase-free water (ISC Bioexpress) and the phases were separated again as described.  This second aqueous phase was added to the first and precipitated with the addition of isopropanol .  The RNA was pelleted, washed once in 75% ethanol, air-dried for 15 minutes and resuspended in ultra-pure RNase-free water.  A DNase reaction was performed as per manufacturer’s instructions (Promega).  Following the DNase reaction, a second organic extraction was performed using TRIzol LS (Invitrogen) as per the manufacturer’s instructions with the following minor modifications: phase-lock heavy gel tubes (Eppendorf) were employed for the phase separation step and the co-precipitant glycoblue (Ambion) was added in the final precipitation step.  The final RNA pellet was resuspended in RNAsecure (Ambion) and quantitated using a spectrophotometer.  A measure of RNA quality was assessed through 260/280 and 260/230 ratios.

treatment_protocol: 50-mL cultures were grown overnight (18h) in 1xPBS/BHI broth.  The cultures were split, pelleted via centrifugation (rcf), and resuspended in neutral (pH 7.4) 1x PBS/BHI broth.  These were incubated for 1 hour at 37oC.  5-mL pellets were collected for subsequent RNA extraction.

value: Environmental, serotype 1/2c, lineage 2,

label: AlexaFluor 555

growth_protocol: All strains were passaged 3 times in BHI at 37oC with moderate shaking for 18 hours.

treatment_protocol: 50-mL cultures were grown overnight (18h) in 1xPBS/BHI broth.  The cultures were split, pelleted via centrifugation (rcf), and resuspended in HCl acidified 1xPBS/BHI broth pH 5.5.  These were incubated for 1 hour at 37oC.  5-mL pellets were collected for subsequent RNA extraction.


title: Spo11-FLAG distribution on chromosome VI in rec8delta cells at meiosis 5 hr

status: Public on May 29 2009

type: genomic

city: Kisarazu

first: Kazuto

last: Kugou

state: Chiba

address: 2-6-7 KazusaKamatatri

department: Department of Frontier Research

institute: Kazusa DNA Research Institute

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI in rec8delta cells at meiosis 5 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome VI)

hyb_protocol: Following denaturation at 100 C for 10 minutes, cooling, and spin-down, 150 micro liter of hybridization mixuter was hybridized for 16 hours at 42 C on GeneChip rikDACF using hybridization oven 640 (affymetrix). Each hybridization mixture contains the end-labbeled DNA, 6xSSPE, 0.005% Triton X-100, 0.1 mg/ml herring sperm DNA, oligo B2 (affymetrix) and hybridization control (affymetrix).

scan_protocol: Washing and scanning protocol (mini-WS1v2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Arrays were scanned by GeneChip Scanner 3000 7G.

data_processing:
Data analyses were performed as previously described in Methods in Enzymology, Elsevier Life Sciences (CA), vol.409,chapter 23,389-410 in “DNA Repair” (edited by J.Campbell) (2006).
Primary data analyses were carried out with GeneChip Operating Software (GCOS 1.4) using default settings to obtain signal and detection p-value.
To discriminate significant signals for binding to DNA, signals from the ChIP fraction scan were compared to the WCE fraction scan with GCOS 1.4 using default settings.

molecule: genomic DNA

source_name: Chromatin-immunoprecipitated DNA

label_protocol: End labeling by terminal transferase

growth_protocol: Cells were grown on a YPD plate for 2 days at 30C. The cells were grown to a density of 4.0x10e7 cells/ml at 30C in SPS presporulation medium (0.5% yeast extract, 1% peptone, 0.17% yeast nitrogen base w/o amino acid and ammonium sulfate, 1% potassium acetate, 0.5% ammonium sulfate, usual level of amino acids, 50 mM potassium phthalate buffer pH 5.0, and antifoam). The cells were suspended in sporulation medium (1% potasium acetate, 1/5 level of amino acids, and polypropylene glycol) to a density of 2x10e7 cells/ml, and incubated at 30C for sporulation.

extract_protocol:
The cells were disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm zirconia beads. Whole cell extract was sonicated to obtain average 500 bp genomic DNA fragments.
The WCE was immunoprecipitated with anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Invitrogen, protein A Dynabeads).
The chromatin immunoprecipitated sample was incubated overnight at 37C with proteinase K, incubated for 6 hrs at 65C, extracted with phenol/chloroform/isoamylalcohol, precipitated, and resuspended in TE and incubated with RNaseA.
The DNA was amplified by PCR after random priming as previously described (Katou et al., 2003, nature). The amplified DNA was digested with Dnase I to a mean size of 100 bp. After DNase I inactivation at 95ºC, fragments were labelled with indicated label protocol.

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 5 hrs in sporulation medium.
The culture was fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

tag: SK1 background strain (rec8delta) expressing FLAG tagged Spo11 protein

tag: Culture

value: 5 hrs after transfer to sporulation medium


title: Spo11-FLAG distribution on chromosome VI in rec8delta cells at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI in rec8delta cells at meiosis 2 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome VI)

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 2 hrs in sporulation medium.
The culture was fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

value: 2 hrs after transfer to sporulation medium


title: DSB sites on chromosome VI in rec8delta cells at meiosis 7 hr

description:
ChIP-chip data used for the mapping of DSB sites on chromosome VI in rec8delta rad50S mutant cells at meiosis 7 hr
Used baseline: WCE fraction (SPO11-FLAG rad50S rec8delta cells at meiosis 7 hr, chromosome VI)

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 7 hrs in sporulation medium.
The cells were washed with sterilized water. The cells were washed with ice-cold TBS, freezed with liquid nitrogen, and stored at -80C.

tag: SK1 background strain (rec8delta rad50S mutant) expressing FLAG tagged Spo11 protein

value: 7 hrs after transfer to sporulation medium


title: Spo11-FLAG distribution on chromosome VI in rec8delta cells at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI in rec8delta cells at meiosis 1.5 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome VI)

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 1.5 hrs in sporulation medium.
The culture was fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

value: 1.5 hrs after transfer to sporulation medium


title: Spo11-FLAG distribution on chromosome VI in rec8delta cells at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI in rec8delta cells at meiosis 4 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome VI)

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 4 hrs in sporulation medium.
The culture was fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

value: 4 hrs after transfer to sporulation medium


title: DSB sites on chromosome VI at meiosis 7 hr

description:
ChIP-chip data used for the mapping of DSB sites on chromosome VI in rad50S mutant cells at meiosis 7 hr
Used baseline: WCE fraction (SPO11-FLAG rad50S cells at meiosis 7 hr, chromosome VI)

tag: SK1 background strain (rad50S mutant) expressing FLAG tagged Spo11 protein


title: Spo11-FLAG distribution on chromosome VI at 4 hrs in sporulation medium  containing HU

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at 4 hrs in sporulation medium containing HU
Used baseline: WCE fraction (SPO11-FLAG cells at 4 hrs in SPM containing HU, chromosome VI)

scan_protocol: Washing and scanning protocol (mini-WS1v2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Arrays were scanned by Hewlett-Packard GeneArray Scanner at an emission wavelength of 560 nm at 7.5 microM resolution.

growth_protocol: Cells were grown on a YPD plate for 2 days at 30C. The cells were grown to a density of 4.0x10e7 cells/ml at 30C in SPS presporulation medium (0.5% yeast extract, 1% peptone, 0.17% yeast nitrogen base w/o amino acid and ammonium sulfate, 1% potassium acetate, 0.5% ammonium sulfate, usual level of amino acids, 50 mM potassium phthalate buffer pH 5.0, and antifoam). The cells were suspended in sporulation medium (1% potasium acetate, 1/5 level of amino acids, and polypropylene glycol) to a density of 2x10e7 cells/ml, and incubated at 30C for sporulation. HU stock solution (1 M HU dissolved in sporulation medium) was added to a final concentration of 100 mM at 15 min after transfer to the sporulation medium.

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 4 hrs in sporulation medium containing HU.
The cells were washed with SPM, resuspended in 100 ml of SPM, fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

tag: SK1 background strain expressing FLAG tagged Spo11 protein

value: 4 hrs in sporulation medium containing 100 mM HU


title: Spo11-FLAG distribution on chromosome VI in rec8delta cells at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI in rec8delta cels at meiosis 3 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome VI)

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 3 hrs in sporulation medium.
The culture was fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

tag: Strain

value: 3 hrs after transfer to sporulation medium


title: Spo11-FLAG distribution on chromosome VI at 2 hrs in sporulation medium containing HU

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at 2 hrs in sporulation medium containing HU
Used baseline: WCE fraction (SPO11-FLAG cells at 4 hrs in SPM containing HU, chromosome VI)

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 2 hrs in sporulation medium containing HU.
The cells were washed with SPM, resuspended in 100 ml of SPM, fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

value: 2 hrs in sporulation medium containing 100 mM HU


title: Rec8-FLAG distribution on chromosome VI at meiosis 5 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome VI at meiosis 5 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)

tag: SK1 background strain expressing FLAG tagged Rec8 protein


title: WCE fraction (SPO11-FLAG cells at 4 hrs in sporulation medium containing HU, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Spo11-FLAG distribution on chromosome VI at 2 and 4 hrs in SPM containing HU

data_processing:
Data analyses were performed as previously described in Methods in Enzymology, Elsevier Life Sciences (CA), vol.409,chapter 23,389-410 in “DNA Repair” (edited by J.Campbell) (2006).
Primary data analyses were carried out with GeneChip Operating Software (GCOS 1.4) using default settings to obtain signal and detection p-value.

source_name: Fragmented whole genomic DNA

extract_protocol:
The cells were disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm zirconia beads. Whole cell extract was sonicated to obtain average 500 bp genomic DNA fragments.
0.7% of the WCE was used for a preparation of WCE fraction (input).
The WCE fraction were incubated overnight at 37C with proteinase K, incubated for 6 hours at 65C, extracted with phenol/chloroform/isoamylalcohol, precipitated, and resuspended in TE and incubated with RNaseA.
The DNA was amplified by PCR after random priming as previously described (Katou et al., 2003, nature). The amplified DNA was digested with Dnase I to a mean size of 100 bp. After DNase I inactivation at 95ºC, fragments were labelled with indicated label protocol.


title: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Spo11-FLAG distribution on chromosome VI in rec8delta cells at meiosis 1.5-5 hr


title: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Rec8-FLAG distribution on chromosome VI at meiosis 1.5-5 hr


title: WCE fraction (SPO11-FLAG rad50S cells at meiosis 7 hr, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of DSB sites on chromosome VI at meiosis 7 hr


title: WCE fraction (SPO11-FLAG rad50S rec8delta cells at meiosis 7 hr, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of DSB sites on chromosome VI in rec8delta cells at meiosis 7 hr


title: WCE fraction (MRE11-FLAG cells at meiosis 4.5 hr, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Mre11-FLAG distribution on chromosome VI at meiosis 1.5-5 hr

extract_protocol:
The cells were disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm glass beads. Whole cell extract was sonicated to obtain average 500 bp genomic DNA fragments.
0.7% of the WCE was used for a preparation of WCE fraction (input).
The WCE fraction were incubated overnight at 37C with proteinase K, incubated for 6 hours at 65C, extracted with phenol/chloroform/isoamylalcohol, precipitated, and resuspended in TE and incubated with RNaseA.
The DNA was amplified by PCR after random priming as previously described (Katou et al., 2003, nature). The amplified DNA was digested with Dnase I to a mean size of 100 bp. After DNase I inactivation at 95ºC, fragments were labelled with indicated label protocol.

treatment_protocol:
100 ml culture (2x10e9 cells) was taken at 4.5 hrs in sporulation medium.
The culture was fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.

tag: SK1 background strain expressing FLAG tagged Mre11 protein

value: 4.5 hrs after transfer to sporulation medium


title: WCE fraction (SPO11-FLAG cells at meiosis 4.5 hr, chromosome VI)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Spo11-FLAG distribution on chromosome VI at meiosis 1.5-5 hr


title: WCE fraction (SPO11-FLAG rad50S rec8delta cells at meiosis 7 hr, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of DSB sites on chromosome III-V in rec8delta cells at meiosis 7 hr

hyb_protocol: Following denaturation at 100 C for 10 minutes, cooling, and spin-down, 200 micro liter of hybridization mixuter was hybridized for 16 hours at 42 C on GeneChip SC3456a520015F using hybridization oven 640 (affymetrix). Each hybridization mixture contains the end-labbeled DNA, 6xSSPE, 0.005% Triton X-100, 0.1 mg/ml herring sperm DNA, oligo B2 (affymetrix) and hybridization control (affymetrix).

scan_protocol: Washing and scanning protocol (EukGE-WS1v3) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Arrays were scanned by GeneChip Scanner 3000 7G.


title: WCE fraction (SPO11-FLAG cells at 4 hrs in sporulation medium containing HU, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Spo11-FLAG distribution on chromosome III-V at 2 and 4 hrs in SPM containing HU

scan_protocol: Washing and scanning protocol (EukGE-WS1v3) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Arrays were scanned by Hewlett-Packard GeneArray Scanner at an emission wavelength of 560 nm at 7.5 microM resolution.


title: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Spo11-FLAG distribution on chromosome III-V in rec8delta cells at meiosis 1.5-5 hr


title: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Rec8-FLAG distribution on chromosome III-V at meiosis 1.5-5 hr


title: WCE fraction (SPO11-FLAG rad50S cells at meiosis 7 hr, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of DSB sites on chromosome III-V at meiosis 7 hr


title: WCE fraction (SPO11-FLAG cells at meiosis 4 hr, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Spo11-FLAG distribution on chromosome III-V at meiosis 1.5-5 hr


title: WCE fraction (MRE11-FLAG cells at meiosis 4 hr, chromosome III-V)

description:
WCE fraction used for the nomalization of ChIP fraction
Baseline of Mre11-FLAG distribution on chromosome III-V at meiosis 1.5-5 hr


title: Benidipine vs. Control

status: Public on Jun 16 2009

city: Asahi-machi, Machida-shi

first: Ayako

last: Kawabata

state: Tokyo

department: BioFrontier Laboratories

institute: Kyowa Hakko Kogyo co.,LTD.

description: compared high-salt plus benidipine and high-salt plus vehicle treated rat

hyb_protocol: Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential

scan_protocol: Scanned on ScanArray 4000 scanner (GSI Lumonics) Images were quantified using QuantArray (GSI Lumonics).

data_processing: Intensity and background intensity of ch1 and ch2 were imported to GeneSpring V6. LOWESS normalization were performed in GeneSpring. Normalized log2 ratio were calculated by the equation LOG (normalized ratio, 2) using EXCEL.

organism: Rattus norvegicus

source_name: Total RNA from hearts of high-salt fed rats labeled with Cyanine-5 (red).

label_protocol: Cy3- or Cy5-labeled target synthesized from 500ng of total RNA using Agilent Low RNA Input Fluorescent Linear Amplification Kit following manufacture's instructions

extract_protocol: Total RNA extracted using Trizol following manufacturer's instructions

tag: Dahl salt-sensitive rat

tag: Age

value: 21 weeks

tag: fed with high salt (FR-2 containing 8% NaCl) diet

tag: Tissue

value: heart

source_name: Total RNA from hearts of high-salt + benidipine treated rats labeled with Cyanine-3 (green).

tag: treated with high salt (8% NaCl) + Benidipine


title: Listeria monocytogenes LMB0622_bile stress_rep 1

description:
Slide Bar Code: 126456
There were three distinct stresses: acid adaptation (pH5.5); acid stress (pH 3.5); bile stress (pH 5.5/0.3% Bile).
All strains underwent 3 biological replicates (rep 1, 2, or 3). Biological replicates that did not correlate with each other (pearson correlation >0.6) were discarded from downstream analysis.

hyb_protocol: The labeled cDNA pellets were resuspended in 70uL of hybridization buffer (5x denhardt’s/4x SSC) and boiled for 2 minutes. Samples were centrifuged for 1 minute and applied to the slides under Erie Scientific Lifter slips (24x60mM). Hybridizations were conducted in a 45oC water bath in corning hybridization chambers for 18 hours. Post hybridization washes consisted of three 1 minute TNT (Tris/NaCl/Tween) washes (gently shaking). The slides were dried using a Microarray High-Speed Centrifuge (TeleChem International, Inc).

data_processing: TIFF images were processed using SPOTfinder software (TIGR TM4 software suite) using the otsu spot setting and employing background subtraction within the spotfinding software. All apot intensities for spots labelled "reserved", "obsolete", or "empty" were discarded prior to normalization.  The resultant median intensities were imported into MATlab software (Mathworks, Inc) for lowess normalization. The normalized intensities and ratios were then imported into an access database. Spot quality control filtering was conducted in access (Microsoft) and consisted of the following criterion: 1) Signal:Noise ratio must be greater than 2 (A488) or 10 (A555); 2) The mean spot intensity must be within 25% of the median spot intensity; 3) The coefficient of variance for replicate spots must be less than 0.2; 4)Those spots with only a single channel passing the previous three filters were then low-intensity filtered with a cutoff of 4000.

label_protocol:
cDNA was generated as follows: 3μg of total RNA and 150ng of random hexamers (RHs) were combined and heated to 70oC for 10 minutes. This RNA:RHs mixture was snap cooled on ice for 1 minute. The remaining reagents from the SuperScript III First Strand Synthesis Kit® (Invitrogen) were added to the following final concentrations: 1.2x RT Buffer; 6.1mM MgCl2; 12mM DTT; 2.7mM dNTP’s (Promega) which contained aa-dUTP (Ambion) at a ratio of 3.5:1; 40U RNaseOUT; and 760U SuperScript III. This mixture was incubated for 18h at 42oC. Following the 42oC incubation, EDTA (pH 8.0) and NaOH were added to a final concentration of 0.1M and 0.2M, respectively. This mixture was incubated at 65oC for 15 minutes in order to hydrolyze the RNA. The solution was neutralized by the addition of 1M Tris-HCl pH 7.0 to a final concentration of 0.3M. The cDNA was cleaned up using a QiaQuick PCR clean-up column (Qiagen) with the following modifications: all spins except the elution step were conducted at 6,000 rpms and the column drying spin (step 6) was extended to 5 minutes; the wash buffer and elution buffers consisted of 5mM KPO4/80% EtOH and 4mM KPO4, respectively, to avoid Tris contamination in the cDNA; finally, the elution buffer was preheated to 37oC and the column was incubated at 37oC for 5 minutes prior to elution from the column. The eluted cDNA was dried down in a centrivap for 45 minutes at 40oC. The resultant pellet was either stored at -20oC until later use or resuspended in 5ul of water and incubated for 2-3 hours at 55oC in preparation for a labeling reaction.
The labeling reactions were performed utilizing AlexaFluor 555 and AlexaFluor 488 as per manufacturer’s instructions with the minor modification of an overnight incubation (instead of 1 hour). Labeling reactions were cleaned up as described above for cDNA clean-up, with the replacement of the KPO4 buffers with manufacturer supplied buffers. The eluted labeled cDNA was quantified on a spectrophotometer. cDNA concentrations were calculated and combined such that the same amount of cDNA was used for each condition (control vs. stress). Each hybridization consisted of between 2.4 – 4.6nM cDNA (per condition) and 60 – 480 pmol dye incorporation (per dye)

growth_protocol: All strains were passaged 3 times in BHI at 37oC with moderate shaking for 18 hours prior to all stress protocols.

extract_protocol: RNA was extracted using TRIzol (Invitrogen) as per the manufacturer’s instructions with only minor modifications. Briefly, bacterial pellets were resuspended in 3-mL of TRIzol followed by the addition of 0.1mm Zirconia beads (BIOSPEC Products Inc) for cell disruption. This mixture underwent rapid horizontal vortexing for 1 hour at 4oC. The beads were pelleted out by centrifugation and chloroform was added for organic extraction. Phase separation was achieved through centrifugation at which time the aqueous phase was removed to a separate tube. The organic phase was back-extracted by the addition of ultra-pure RNase-free water (ISC Bioexpress) and the phases were separated again as described. This second aqueous phase was added to the first and precipitated with the addition of isopropanol . The RNA was pelleted, washed once in 75% ethanol, air-dried for 15 minutes and resuspended in ultra-pure RNase-free water. A DNase reaction was performed as per manufacturer’s instructions (Promega). Following the DNase reaction, a second organic extraction was performed using TRIzol LS (Invitrogen) as per the manufacturer’s instructions with the following minor modifications: phase-lock heavy gel tubes (Eppendorf) were employed for the phase separation step and the co-precipitant glycoblue (Ambion) was added in the final precipitation step. The final RNA pellet was resuspended in RNAsecure (Ambion) and quantitated using a spectrophotometer. A measure of RNA quality was assessed through 260/280 and 260/230 ratios.

treatment_protocol: 50-mL cultures were grown overnight (18h) in 1xPBS/BHI broth. The cultures were split, pelleted via centrifugation, and resuspended in a netral pH (pH 7.4) 1x PBS/BHI broth. These were incubated for 1 hour at 37oC. The culture was then pelleted via centrifugation and resuspended in a neutral pH (pH 7.4) 1x PBS/BHI broth.  This was incubated for 1 hour at 37oC.  Lastly, the culture was pelleted via centrifugation, and resuspended in a sub-lethal pH (pH 5.5) 1xPBS/BHI broth.  This was incubated for 1 hour at 37oC.  A 5mL aliquot was subsequently pelleted for RNA extraction.

label_protocol:
cDNA was generated as follows: 3μg of total RNA and 150ng of random hexamers (RHs) were combined and heated to 70oC for 10 minutes. This RNA:RHs mixture was snap cooled on ice for 1 minute. The remaining reagents from the SuperScript III First Strand Synthesis Kit® (Invitrogen) were added to the following final concentrations: 1.2x RT Buffer; 6.1mM MgCl2; 12mM DTT; 2.7mM dNTP’s (Promega) which contained aa-dUTP (Ambion) at a ratio of 3.5:1; 40U RNaseOUT; and 760U SuperScript III. This mixture was incubated for 18h at 42oC. Following the 42oC incubation, EDTA (pH 8.0) and NaOH were added to a final concentration of 0.1M and 0.2M, respectively. This mixture was incubated at 65oC for 15 minutes in order to hydrolyze the RNA. The solution was neutralized by the addition of 1M Tris-HCl pH 7.0 to a final concentration of 0.3M. The cDNA was cleaned up using a QiaQuick PCR clean-up column (Qiagen) with the following modifications: all spins except the elution step were conducted at 6,000 rpms and the column drying spin (step 6) was extended to 5 minutes; the wash buffer and elution buffers consisted of 5mM KPO4/80% EtOH and 4mM KPO4, respectively, to avoid Tris contamination in the cDNA; finally, the elution buffer was preheated to 37oC and the column was incubated at 37oC for 5 minutes prior to elution from the column. The eluted cDNA was dried down in a centrivap for 45 minutes at 40oC. The resultant pellet was either stored at -20oC until later use or resuspended in 5uL of water and incubated for 2-3 hours at 55oC in preparation for a labeling reaction.
The labeling reactions were performed utilizing AlexaFluor 555 and AlexaFluor 488 as per manufacturer’s instructions with the minor modification of an overnight incubation (instead of 1 hour). Labeling reactions were cleaned up as described above for cDNA clean-up, with the replacement of the Phosphate buffers with manufacturer supplied buffers. The eluted labeled cDNA was quantified on a spectrophotometer. cDNA concentrations were calculated and combined such that the same amount of cDNA was used for each condition (control vs. stress). Each hybridization consisted of between 2.4 – 4.6nM cDNA (per condition) and 60 – 480 pmol dye incorporation (per dye)

treatment_protocol: 50-mL cultures were grown overnight (18h) in 1xPBS/BHI broth. The cultures were split, pelleted via centrifugation, and resuspended in a sub-lethal pH (pH 5.5) 1x PBS/BHI broth. These were incubated for 1 hour at 37oC. The culture was then pelleted via centrifugation and resuspended in a lethal pH (pH 3.5) 1x PBS/BHI broth.  This was incubated for 1 hour at 37oC.  Lastly, the culture was pelleted via centrifugation, and resuspended in a sub-lethal pH (pH 5.5) 1xPBS/BHI broth that contained 0.3% BILE.  This was incubated for 1 hour at 37oC.  The entire 10mL culture was subsequently pelleted for RNA extraction.


title: Control vs. Normal

description: compared high salt plus vehicle treated rat and normal fed rat

source_name: Total RNA from hearts of normal fed rats labeled with Cyanine-5 (red).

tag: fed with normal (FR-2 containing 0.19% NaCl) diet

source_name: Total RNA from hearts of high salt fed rats labeled with Cyanine-3 (green).

tag: fed with high salt (8% NaCl) diet


title: DSB sites on chromosome III-V in rec8delta cells at meiosis 7 hr

description:
ChIP-chip data used for the mapping of DSB sites on chromosome III-V in rec8delta rad50S mutant cells at meiosis 7 hr
Used baseline: WCE fraction (SPO11-FLAG rad50S rec8delta cells at meiosis 7 hr, chromosome III-V)

extract_protocol:
The cells were disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm zirconia beads. Whole cell extract was sonicated to obtain average 500 bp genomic DNA fragments.
The WCE was immunoprecipitated with anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Invitrogen, protein A Dynabeads).
The chromatin immunoprecipitated sample was incubated overnight at 65ºC, incubated with proteinase K, extracted with phenol/chloroform/isoamylalcohol, precipitated, and resuspended in TE and incubated with RNaseA.
The DNA was amplified by PCR after random priming as previously described (Katou et al., 2003, nature). The amplified DNA was digested with Dnase I to a mean size of 100 bp. After DNase I inactivation at 95ºC, fragments were labelled with indicated label protocol.


title: Rec8-FLAG distribution on chromosome VI at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome VI at meiosis 4 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)


title: Rec8-FLAG distribution on chromosome VI at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome VI at meiosis 1.5 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)


title: Mre11-FLAG distribution on chromosome VI at meiosis 5 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome VI at meiosis 5 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4.5 hr, chromosome VI)

extract_protocol:
The cells were disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm glass beads. Whole cell extract was sonicated to obtain average 500 bp genomic DNA fragments.
The WCE was immunoprecipitated with anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Invitrogen, protein A Dynabeads).
The chromatin immunoprecipitated sample was incubated overnight at 37C with proteinase K, incubated for 6 hrs at 65C, extracted with phenol/chloroform/isoamylalcohol, precipitated, and resuspended in TE and incubated with RNaseA.
The DNA was amplified by PCR after random priming as previously described (Katou et al., 2003, nature). The amplified DNA was digested with Dnase I to a mean size of 100 bp. After DNase I inactivation at 95ºC, fragments were labelled with indicated label protocol.


title: Mre11-FLAG distribution on chromosome VI at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome VI at meiosis 2 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Mre11-FLAG distribution on chromosome VI at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome VI at meiosis 1.5 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Mre11-FLAG distribution on chromosome VI at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome VI at meiosis 4 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Spo11-FLAG distribution on chromosome VI at meiosis 5 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at meiosis 5 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Mre11-FLAG distribution on chromosome VI at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome VI at meiosis 3 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Rec8-FLAG distribution on chromosome VI at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome VI at meiosis 3 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)


title: Spo11-FLAG distribution on chromosome VI at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at meiosis 2 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Spo11-FLAG distribution on chromosome VI at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at meiosis 3 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Spo11-FLAG distribution on chromosome VI at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at meiosis 1.5 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Rec8-FLAG distribution on chromosome VI at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome VI at meiosis 2 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)


title: DSB sites on chromosome III-V at meiosis 7 hr

description:
ChIP-chip data used for the mapping of DSB sites on chromosome III-V in rad50S mutant cells at meiosis 7 hr
Used baseline: WCE fraction (SPO11-FLAG rad50S cells at meiosis 7 hr, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V in rec8delta cells at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V in rec8delta cells at meiosis 4 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V in rec8delta cells at meiosis 5 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V in rec8delta cells at meiosis 5 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V in rec8delta cells at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V in rec8delta cells at meiosis 2 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V at 2 hrs in sporulation medium containing HU

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V at 2 hrs in sporulation medium containing HU
Used baseline: WCE fraction (SPO11-FLAG cells at 4 hrs in SPM containing HU, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V in rec8delta cells at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V in rec8delta cels at meiosis 3 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V in rec8delta cells at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V in rec8delta cells at meiosis 1.5 hr
Used baseline: WCE fraction (SPO11-FLAG rec8delta cells at meiosis 3 hrs, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V at 4 hrs in sporulation medium containing HU

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V at 4 hrs in sporulation medium containing HU
Used baseline: WCE fraction (SPO11-FLAG cells at 4 hrs in SPM containing HU, chromosome III-V)


title: Rec8-FLAG distribution on chromosome III-V at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome III-V at meiosis 3 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome III-V)


title: Mre11-FLAG distribution on chromosome III-V at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome III-V at meiosis 2 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Mre11-FLAG distribution on chromosome III-V at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome III-V at meiosis 4 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Mre11-FLAG distribution on chromosome III-V at meiosis 5 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome III-V at meiosis 5 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Rec8-FLAG distribution on chromosome III-V at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome III-V at meiosis 1.5 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome III-V)


title: Mre11-FLAG distribution on chromosome III-V at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome III-V at meiosis 3 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Rec8-FLAG distribution on chromosome III-V at meiosis 2 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome III-V at meiosis 2 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome III-V)


title: Spo11-FLAG distribution on chromosome VI at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome VI at meiosis 4 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4.5 hr, chromosome VI)


title: Mre11-FLAG distribution on chromosome III-V at meiosis 1.5 hr

description:
ChIP-chip data used for the mapping of Mre11 distribution on chromosome III-V at meiosis 1.5 hr
Used baseline: WCE fraction (MRE11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Rec8-FLAG distribution on chromosome III-V at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Rec8 distribution on chromosome III-V at meiosis 4 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V at meiosis 5 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V at meiosis 5 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V at meiosis 4 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V at meiosis 4 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: Spo11-FLAG distribution on chromosome III-V at meiosis 3 hr

description:
ChIP-chip data used for the mapping of Spo11 distribution on chromosome III-V at meiosis 3 hr
Used baseline: WCE fraction (SPO11-FLAG cells at meiosis 4 hr, chromosome III-V)


title: RH2_rpd3_60 min

status: Public on May 21 2009

city: Madison

first: Audrey

middle: P.

last: Gasch

state: WI

address: 425-g Henry Mall

department: Laboratory of Genetics

institute: University of Wisconsin Madison

description: Comparison of wt before and after reciprocal heat shock from 37C to 25C.  Cells grown ~3 generations in YPD at 37C before centrifugation and resuspension in fresh YPD at 25C - replicate #2

hyb_protocol: As indicated in Gasch AP Methods in Enzymology 350: 393-414  To prepare the probe for hybridization to a ~22 x 22 mm microarray, combine 5/zl of each of the Cy-dUTP labeled samples or 10/zl of the combined aminoallyllabeled sample, 1/zl of 10 mg/ml poly(A) carrier (Sigma), 2.3/zl 20× SSC, and 0.36 tzl 10% SDS for a final volume of 14/zl (if the microarray is larger than 22 x 22 mm add to the 10 #1 of probe solution 2.6 #1 poly(A), 5.0 20x SSC, and 0.78 #1 10% SSD and adjust the final volume to 30 #1 with water). Denature the probe by heating at 94 ° for 3 min, then cool to room temperature by rapidly transferring to a microfuge and spinning for 10 sec (avoid adding bubbles during handling). Do not chill the probe on ice as salt will precipitate from the solution. Prepare the hybridization chamber (Coming): place the microarray into the chamber and add ~20/zl 3× SSC to each of the hybridization wells in the chamber and an additional -~30/zl of 3x SSC divided into isolated drops on the edges of the microarray. Insufficient volume will result in inadequate chamber hydration during hybridization, causing the probe to dry out under the coverslip. Select multiple microscope slide coverslips of the appropriate size (Coming) that appear free of dust, chips, and scratches, and prop them for easy access with a tweezers. Adding the probe to the microarray and sealing with the coverslip requires some experience and personal technique. It is advisable to practice the following procedure before hand. To the center of the microarray, deposit 12-15/zl of the denatured and cooled probe for 22 x 22 mm arrays, or 28-30 #1 probe for 22 x 44 mm microarrays. Bubbles can prevent proper hybridization, and therefore they should be avoided and removed when possible. Large bubbles can be popped with the comer of a coverslip, but great care should be taken not to touch the microarray surface. Quickly place the coverslip directly over the deposited probe and gently drop or place the coverslip over the array. The coverslip should not be moved so as to prevent scratching of the microarray surface; however, in unavoidable cases it can be gently positioned by sliding. Close the hybridization chamber and gently and quickly submerge the chamber in a 65 ° water bath. For optimal signal, hybridize the arrays for 7-16 hr. For consistent results, the hybridization conditions, including hybridization time, should be maintained for all microarrays.

scan_protocol: Arrays were scanned on an Axon 4000B scanner

data_processing: Data were extracted using GenePix 6.0.  Data were normalized by regional mean-centering as described by Lyne et al. 2003 BMC Genomics

label: Oyster 550 cyanine dye

source_name: BYrpd3 grown at 37C (time 0 reference)

label_protocol: Indirect labeling using amino-allyl-dUTP and cyanine dyes from Flownamics (Madison, Wi) as described in Gasch AP Methods in Enzymology 350: 393-414

growth_protocol: Unless otherwise noted, cells were grown 3 doublings in YPD or specified medium at 30C to OD600 ~0.5

extract_protocol: Hot-phenol RNA extraction as described in Gasch AP Methods in Enzymology 350: 393-414

treatment_protocol: Stresses were added directly to the culture at the indicated concentrations.  For heat shock experiments, cells were collected by rapid filtration at 25C and resuspended in fresh YPD heated to 37C.  For reverse heat shock experiments, cells were grown 3 doublings in YPD at 37C, collected by brief centrifugation, and resuspended in fresh YPD at 25C

tag: Strain BY4741-rpd3:KanMX (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 rpd3::KanMX)

label: Oyster cyanine 650 dye

source_name: BYrpd3 60 min after shift to 25C YPD


title: RH2_wt_60 min

source_name: BY4741 grown at 37C (time 0 reference)

tag: Strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0)

source_name: BY4741 60 min after shift to 25C YPD


title: RH2_wt_40 min

source_name: BY4741 40 min after shift to 25C YPD


title: RH2_rpd3_40 min

source_name: BYrpd3 40 min after shift to 25C YPD


title: RH2_rpd3_10 min

source_name: BYrpd3 10 min after shift to 25C YPD


title: RH_1_rpd3_5 min

description: Comparison of rpd3 before and after reciprocal heat shock from 37C to 25C.  Cells grown ~3 generations in YPD at 37C before centrifugation and resuspension in fresh YPD at 25C - replicate #2

source_name: BYrpd3 5 min after shift to 25C YPD


title: RH_1_wt_10 min

description: Comparison of wt before and after reciprocal heat shock from 37C to 25C.  Cells grown ~3 generations in YPD at 37C before centrifugation and resuspension in fresh YPD at 25C - replicate #1

source_name: BY4741 10 min after shift to 25C YPD


title: NaCl2_wt_45 min

description: Comparison of wt before and after addition of NaCl to 0.7M in YPD - replicate #2

source_name: BY4741 45 min after addition of NaCl to 0.75M

source_name: BY4741 grown in YPD at 30C (time 0 reference)


title: NaCl2_wt_60 min

source_name: BY4741 60 min after addition of NaCl to 0.75M


title: RH2_wt_10 min


title: RH_1_rpd3_10 min


title: NaCl2_rpd3_45 min

description: Comparison of rpd3 before and after addition of NaCl to 0.7M in YPD - replicate #2

source_name: BYrpd3 45 min after addition of NaCl to 0.75M

source_name: BYrpd3 grown in YPD at 30C (time 0 reference)


title: RH_1_rpd3_20 min

source_name: BYrpd3 20 min after shift to 25C YPD


title: NaCl2_rpd3_60 min

source_name: BYrpd3 60 min after addition of NaCl to 0.75M


title: NaCl2_rpd3_30 min

source_name: BYrpd3 30 min after addition of NaCl to 0.75M