title: Patient_65_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_65, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:2, ER:2, PR:4, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:2.5, bcl2_Score:0


title: Patient_44_Cy5

status: Public on Jan 21 2006

type: RNA

city: Heidelberg

first: Olaf

last: Thuerigen

address: Im Neuenheimer Feld 280

department: Molecular Genetics (B060)

country: Germany

institute: German Cancer Research Center

description: aRNA produced by T7 amplification was reversely transcribed and subsequently labeled using Klenow fragment and Cy-dUTPs. Hybridization was carried out at 37 °C in UltraHyb buffer (Ambion) on a GeneTAC hybridization station (Genomic Solutions).

data_processing: spot replicates were averaged. Spots not recognized by the image analysis software "GenePix Pro 5.0" (Axon Instruments) were excluded from the calculations. Raw fluorescence intensities were normalized applying the variance stabilization method (W. Huber et al., Bioinformatics 18 Suppl. 1, 2002). To eliminate low quality data, the data points were ranked according to spot homogeneity, as assayed by the ratio of median to mean fluorescence intensity, the ratio of spot to local background intensity and the standard deviation of the log2 ratios between spot replicates. Those data points ranked among the lower 20% were removed from the data set. Genes that could not be quantified in more than 33% of all experiments of the experimental series after filtering were excluded as well.

molecule: total RNA

organism: Homo sapiens

source_name: 2µg universal human reference total RNA, pooled from 10 human cell lines representing distinct tissues (Stratagene #740000), amplified and labeled using TAcKLE Protocol (PMID: 15718295).

tag: Grading

value: NA, HER2:NA, ER:NA, PR:NA, Therapy_Status_Pathological:NA, ypN:NA, cT_max:NA, bcl2_Score:NA

source_name: 2µg breast tissue total RNA from patient Patient_44, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G2, HER2:0, ER:8, PR:4, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:6, bcl2_Score:1


title: Patient_42_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_42, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G2, HER2:1, ER:6, PR:6, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:3.2, bcl2_Score:3


title: Patient_41_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_41, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:0, ER:0, PR:0, Therapy_Status_Pathological:pPR, ypN:N+, cT_max:3, bcl2_Score:0


title: Patient_39_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_39, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:3, ER:0, PR:0, Therapy_Status_Pathological:pCR, ypN:N0, cT_max:5.5, bcl2_Score:1


title: Patient_26_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_26, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:0, ER:0, PR:0, Therapy_Status_Pathological:pCR, ypN:N0, cT_max:3.9, bcl2_Score:0


title: Patient_62_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_62, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G2, HER2:0, ER:0, PR:0, Therapy_Status_Pathological:pCR, ypN:N0, cT_max:3, bcl2_Score:0


title: Patient_64_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_64, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G2, HER2:0, ER:12, PR:12, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:2.6, bcl2_Score:3


title: Patient_65_Cy5


title: Patient_44_Cy3


title: Patient_62_Cy3


title: Patient_72_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_72, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: no data, HER2:0, ER:8, PR:4, Therapy_Status_Pathological:pNC, ypN:N0, cT_max:3.5, bcl2_Score:1


title: Patient_51_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_51, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G2, HER2:0, ER:12, PR:12, Therapy_Status_Pathological:pPR, ypN:N+, cT_max:4.5, bcl2_Score:0


title: Patient_69_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_69, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G2, HER2:0, ER:no data, PR:6, Therapy_Status_Pathological:pNC, ypN:N0, cT_max:5.5, bcl2_Score:0


title: Patient_77_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_77, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:0, ER:2, PR:0, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:3.1, bcl2_Score:0


title: Patient_77_Cy3


title: Patient_64_Cy5


title: Patient_852_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_852, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: no data, HER2:0, ER:12, PR:12, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:4, bcl2_Score:2

value: NA, HER2:NA, ER:NA, PR:NA, Therapy_Status_Pathological:no data, ypN:NA, cT_max:NA, bcl2_Score:NA


title: Patient_40_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_40, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:0, ER:12, PR:12, Therapy_Status_Pathological:pPR, ypN:N+, cT_max:3.3, bcl2_Score:3


title: testicle.1

status: Public on Jan 19 2006

city: Santa Cruz

first: Charles

last: Sugnet

state: CA

address: Sinsheimer Labs

department: MCD

country: USA

institute: UC Santa Cruz

description: Tissue

hyb_protocol: Targets were hybridized to chips in 7% DMSO solution for 16 hrs overnight at 50. Microarrays were washed, processed with anti-biotin antibodies and streptavidin-phycoerythrin according to the standard Affymetrix protocol.

scan_protocol: standard Affymetrix procedures

data_processing: Intensity values from the DNA arrays were normalized using a quantile normalization (Bolstad et al., 2003), and probe set summaries were derived using the Robust Multi-chip Analysis (RMA) procedure (Irizarry et al., 2003a; Irizarry et al., 2003b) with two modifications.  The first modification was to remove all probes with 17 or more continuous bases that match to any other mouse transcript in order to minimize cross-hybridization issues.  The second modification was to use the mode of the probe intensity values of similar GC content probes for the background estimate of a particular probe.  For example, if a probe has a GC count of 16, then the mode of the intensity of all the probes with a GC count of 16 was used as a background estimate as opposed to RMA in which the mode of all the probes is used as a background estimate for all the probes.

label: biotin

organism: Mus musculus

source_name: testicle

label_protocol: RNA was primed with random hexamers and reverse transcribed. After the reaction was completed, RNA was removed from the reaction by alkaline hydrolysis and the cDNA was purified using Qiagen PCR Quick Purification Kit.  A typical reaction started with 5-6ug of total RNA usually yielded ~3ug of cDNA. The cDNA was then fragmented using DNAseI in an empirically controlled reaction that yields DNA fragments of 50-200 bases. This fragmented cDNA was then end labeled using terminal deoxynucleotidyl transferase and "DNA-Labeling-Reagent-1a (DLR-1a)", which is a biotinylated dideoxynucleoside triphosphate.

extract_protocol: Extracted using the Invitrogen Micro-to-Midi Total RNA Purification System (Catalog # 12183-018). RNA samples were treated with DNAseI for 30 minutes at 37, extracted twice with phenol/chloroform, once with chloroform and ethanol precipitated.

tag: Adult C57/BLJ6 Male Mouse


title: Patient_14_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_14, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:3, ER:0, PR:0, Therapy_Status_Pathological:pCR, ypN:N0, cT_max:9, bcl2_Score:0


title: testicle.3

description: Tissue


title: Patient_51_Cy3


title: Patient_11_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_11, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:0, ER:12, PR:12, Therapy_Status_Pathological:pNC, ypN:N0, cT_max:3, bcl2_Score:2


title: small.intestine.2

description: small

source_name: intestine

tag: Adult C57/BLJ6 Female Mouse


title: spinal.cord.2

description: spinal

source_name: cord


title: spinal.cord.3

description: spinal


title: stomach.2

description: Tissue

source_name: stomach


title: Patient_72_Cy3

source_name: 2µg universal human reference total RNA, pooled from 10 human cell lines representing distinct tissues (Stratagene #740000), amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: NA, HER2:NA, ER:NA, PR:NA, Therapy_Status_Pathological:NA, ypN:NA, cT_max:NA, bcl2_Score:NA


title: skeletal.muscle.1

description: skeletal

source_name: muscle


title: small.intestine.3

description: small


title: stomach.1

description: Tissue


title: spleen.2

description: Tissue

source_name: spleen


title: spleen.3

description: Tissue


title: skeletal.muscle.2

description: skeletal


title: Patient_11_Cy5


title: testicle.2

description: Tissue


title: skeletal.muscle.4

description: skeletal


title: spleen.1

description: Tissue


title: Patient_24_Cy5

source_name: 2µg breast tissue total RNA from patient Patient_24, amplified and labeled using TAcKLE Protocol (PMID: 15718295).

value: G3, HER2:3, ER:9, PR:9, Therapy_Status_Pathological:pPR, ypN:N0, cT_max:3, bcl2_Score:2


title: skeletal.muscle.3

description: skeletal


title: salivary.gland.3

description: salivary

source_name: gland


title: stomach.3

description: Tissue


title: small.intestine.1

description: small


title: salivary.gland.2

description: salivary


title: yDH419 Hmo1 ChIP-chip replicate 2

type: genomic

city: Boston

first: Joseph

last: Wade

state: MA

address: 240 Longwood Ave

department: BCMP

institute: Harvard Medical School

description: ChIP sample value divided by total DNA value

data_processing: Ratio of ChIP sample value to total DNA value

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: yDH419 (Hmo1-3myc) Hmo1 ChIP replicate 2

growth_protocol: YPD growth to OD600 of 0.7

treatment_protocol: Chromatin immunoprecipitation with anti-myc (9E10 monoclonal)

source_name: yDH419 (Hmo1-3myc) total DNA

tag: yDH419 (Hall et al., 2006)

tag: Derivative of BY4742 with 3 myc tags at C-terminus of Hmo1


title: yDH419 Hmo1 ChIP-chip replicate 3

source_name: yDH419 (Hmo1-3myc) ChIP-chip replicate 3


title: pinealgland.2

description: Tissue

source_name: pinealgland


title: salivary.gland.1

description: salivary


title: pinealgland.3

description: Tissue


title: yDH419 Hmo1 ChIP-chip Repeat 1

source_name: yDH419 (Hmo1-3myc) Hmo1 ChIP


title: BY4742 control ChIP-chip

source_name: BY4742 control myc IP

source_name: BY4742 total DNA


title: ovary.3

description: Tissue

source_name: ovary


title: olfactory.bulb.2

description: olfactory

source_name: bulb


title: Patient_69_Cy3

source_name: 2µg breast tissue total RNA from patient Patient_69, amplified and labeled using TAcKLE Protocol (PMID: 15718295).


title: ovary.1

description: Tissue


title: mammarygland.2

description: Tissue

source_name: mammarygland


title: pinealgland.1

description: Tissue


title: lung.1

description: Tissue

source_name: lung


title: olfactory.bulb.3

description: olfactory


title: spinal.cord.1

description: spinal

source_name: cord


title: lung.2

description: Tissue


title: lung.3

description: Tissue


title: kidney.2

description: Tissue

source_name: kidney


title: olfactory.bulb.1

description: olfactory


title: kidney.1

description: Tissue


title: mammarygland.1

description: Tissue


title: ovary.2

description: Tissue


title: mammarygland.3

description: Tissue


title: liver.1

description: Tissue

source_name: liver


title: hind.brain.2

description: hind

source_name: brain


title: liver.2

description: Tissue


title: liver.3

description: Tissue


title: hind.brain.1

description: hind


title: esophagus.3

description: Tissue

source_name: esophagus


title: heart.2

description: Tissue

source_name: heart


title: esophagus.2

description: Tissue


title: kidney.3

description: Tissue


title: heart.1

description: Tissue


title: esophagus.1

description: Tissue


title: cortex.3

description: Tissue

source_name: cortex


title: cortex.1

description: Tissue


title: colon.3

description: Tissue

source_name: colon


title: cerebral.hemisphere.2

description: cerebral

source_name: hemisphere


title: colon.1

description: Tissue


title: PAOI replicate 2 od 0.5

status: Public on Jan 18 2006

city: Seattle

first: Yannick

last: Lequette

state: WA

address: 1959 NE PACiFiC

department: Microbiology HSB -I420 BOX 357242

institute: University of Washington

description: RNA extracted from culture at optical density at 600 nm of 0.5

data_processing: standard Affymetrix procedures

organism: Pseudomonas aeruginosa

source_name: PAOI planktonic culture

tag: PAOI wild type


title: heart.3

description: Tissue


title: cerebral.hemisphere.3

description: cerebral


title: PAOR3 replicate 1 od 0.5

source_name: PAOR3 planktonic culture

tag: PAOI knock-out in qscR gene


title: PAOR3 replicate 2 od 0.5


title: PAOR3 replicate 2 od 0.8

description: RNA extracted from culture at optical density at 600 nm of 0.8


title: PAOI replicate 1 od 0.5


title: cortex.2

description: Tissue


title: cerebellum.3

description: Tissue

source_name: cerebellum


title: cerebellum.2

description: Tissue


title: PAOI replicate 1 od 0.8


title: cerebral.hemisphere.1

description: cerebral


title: cerebellum.1

description: Tissue


title: PAOR3 replicate 2 od 1.4

description: RNA extracted from culture at optical density at 600 nm of 1.4


title: PAOR3 replicate 1 od 1.4


title: colon.2

description: Tissue


title: PAOI replicate 1 od 1.4


title: PAOI replicate 2 od 1.4


title: PAOI replicate 2 od 2.0

description: RNA extracted from culture at optical density at 600 nm of 2.0


title: PAOI replicate 1 od 2.0


title: PAOR3 replicate 1 od 2.0


title: PAOR3 replicate 2 od 3.5

description: RNA extracted from culture at optical density at 600 nm of 3.5


title: PAOR3 replicate 1 od 3.5


title: PAOR3 replicate 2 od 2.0


title: PAOI replicate 2 od 3.5


title: YL117 od 0.5

source_name: YL117 planktonic culture

tag: PAOR3 containing qscR deleted in DNA binding domain under L-arabinose control promoter in attb site


title: YL117 od 1.4


title: YL117 od 3.5


title: YL113 od 0.5

source_name: YL113 planktonic culture

tag: PAOR3 containing qscR under L-arabinose control promoter in attb site


title: YL117 od 0.8


title: YL117 od 2.0


title: YL113 od 0.8


title: YL113 od 1.4

source_name: YL113 plamktonic culture


title: YL113 od 2.0


title: PAOI replicate 1 od 3.5


title: YL113 od 3.5


title: c37_placenta

status: Public on Jan 17 2006

city: Baltimore

first: Erica

last: Allen

state: MD

institute: Johns Hopkins University

description: Array c37 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal.  Dyeswap of array c42.

source_name: 14.5d Pkd1-/- placenta, labeled with Cy3

source_name: 14.5d Pkd1+/+ placenta, labeled with Cy5


title: Day 5 5cc7 T cell blasts, set 1

city: Stanford

first: Morgan

last: Huse

address: 279 Campus Dr

department: Microbiology and Immunology

institute: Stanford

description: RNA was isolated from 5cc7 T cells 5 days after in vitro stimulation with MCC peptide.  This is experiment 1 of 2

data_processing: Normalization using all probe sets scaling

molecule: polyA RNA

source_name: Day 5 5cc7 T cell blasts

tag: 5cc7 T cell receptor transgenic mice, B10A backgorund


title: c88_placenta

description: Array c88 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of arrays c85, c86 and c87.  Replicate of array c84.

source_name: 15.5d Pkd1-/- placenta, labeled with Cy3

source_name: 15.5d Pkd1+/+ placenta, labeled with Cy5


title: hind.brain.3

description: hind

source_name: brain


title: Day 5 5cc7 T cell blasts, set 2

tag: 5cc7 T cell receptor transgenic mice, B10A background


title: c69_placenta

description: Array c69 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c70

source_name: 14.5d Pkd1+/+ placenta, labeled with Cy3

source_name: 14.5d Pkd1-/- placenta, labeled with Cy5


title: c70_placenta

description: Array c70 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c69


title: c40_placenta

description: Array c40 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c36


title: c87_placenta

description: Array c87 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of arrays c84 and c88.  Replicate of arrays c85 and c86.

source_name: 15.5d Pkd1+/+ placenta, labeled with Cy3

source_name: 15.5d Pkd1-/- placenta, labeled with Cy5


title: c42_placenta

description: Array c42 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c37


title: c39_placenta

description: Array c39 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c27


title: c84_placenta

description: Array c84 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of arrays c85, c86 and c87.  Replicate of array c88.


title: c50_placenta

description: Array c50 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c44


title: c85_placenta

description: Array c85 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of arrays c84 and c88.  Replicate of arrays c86 and c87.


title: c44_placenta

description: Array c44 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c50


title: c38_placenta

description: Array c38 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c37-2


title: c37-2_placenta

description: Array c37-2 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c38


title: c27_placenta

description: Array c27 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c39

source_name: 15.5d Pkd1-/- placenta labeled Cy3

source_name: 15.5d Pkd1+/+ placenta labeled Cy5


title: Hippocampus VC mice 2 week off dox 1

status: Public on Jan 12 2006

city: Sant Joan d'Alacant

first: Angel

last: Barco

state: Alicante

address: Av. Santiago Ramón y Cajal

country: Spain

institute: Instituto de Neurociencias (UMH-CSIC)

description: VP16-CREB hippocampus region

hyb_protocol: standard Affymetrix procedures

data_processing: Affymetrix GCOS

source_name: hippocampus

label_protocol: Approximately 0.5 µg of total RNA was processed to produce biotinylated cRNA targets.

extract_protocol: Total RNA was extracted from dissected hippocampi using TRIzol (Invitrogen) according to the manufacturer’s protocol. PolyA mRNA was then isolated using Messagemaker kit (Pharmacia).

tag: 2 week removal of Doxycycline 40 mg/kg, VP16-CREB mice, whole hippocampus


title: Microdissected CA1 region from VC mice 3 weeks off dox 1

description: VP16-CREB hippocampus CA-1 region

source_name: hippocampus CA1 region

tag: 3 weeks removal of Doxycycline 40 mg/kg, VP16-CREB mice, hippocampus CA1 region


title: c86_placenta

description: Array c86 represents cohybridization of 15.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of arrays c84 and c88.  Replicate of arrays c85 and c87.


title: Hippocampus VC mice 2 week off dox 2

description: VP16-CREB hippocampus region


title: Hippocampus VC mice off/on Dox (Reversal for 2 weeks)

description: Wild type hippocampus region

tag: VC mice off/on Dox (Reversal for 2 weeks), Whole hippocampus


title: c36_placenta

description: Array c36 represents cohybridization of 14.5d placentas from a littermate pair of Pkd1 wild-type versus knockout animal. Dyeswap of array c40


title: Hippocampus VC mice 1 week off dox 1

description: VP16-CREB hippocampus region

tag: 1 week removal of Doxycycline 40 mg/kg, VP16-CREB mice, whole hippocampus


title: Plasmacytoid DC2_U74A

status: Public on Jan 11 2006

city: baltimore

first: Drew

last: Pardoll

address: orleans st

institute: Drew Pardoll

description: image analysis of each GeneChip was done through the GeneChip Operating System software from Affymetrix (GCOS1.1.1), using the standard default settings.  We used global scaling for comparisons between different chips, scaling all probe sets to a user defined target intensity (TGT) of 150. To ascertain the quality control of the total RNA from each sample, we used the Agilent Bioanalyzer Lab on a Chip technology, and confirmed the rRNA ratios and clean run patterns of each sample. Likewise, this technology is used to confirm the quality of the RNA in the form of cRNA and fragmented cRNA. To assess QC of the hybridization, GeneChip image, and comparison between chips, we confirmed the following parameters: scaling factor values within comparable range; low background values (between 20 and 100); high percentage of present calls (between 25 and 50); consistent 3’/5’ ratios of Gapdh as representation of housekeeping genes, and presence or absence of Bio B and C as internal spike controls.

hyb_protocol: hybridized 10 μg of total fragmented cRNA to the Affymetrix murine genome GeneChip array U74 set for 16 h at 45 oC with constant rotation (60 rpm).

scan_protocol: Fluorescence was detected using the Affymetrix- GS3000 GeneArray Scanner

label: biotinylation then SA-PE as readout

source_name: Mouse Spleen Plasmacytoid DC

label_protocol: We label anti-sense cRNA using BioArray RNA High Yield Transcript Labelling kit (ENZO Life Sciences Inc).

extract_protocol: Rwe isolated total RNA using the Trizol Reagent (Invitrogen) then purified using the RNeasy Mini Kit (Qiagen). RNA from spleen cDCs, PDCs and IKDCs were processed using the two-round RNA amplification protocol described by Affymetrix (Affymetrix GeneChip Expression Manual Small Sample_2) (http://www.affymetrix.com/support/technical/technotes/smallv2_technote.pdf).

tag: BALB/c, female, 6-8 weeks old, spleen, FACS-sorted plasmacytoid DC, untreated samples (Ex vivo)


title: Plasmacytoid DC1_U74A

tag: BALB/c, female, 6-8 weeks old, spleen, FACS-sorted plasmacytoid DCs, untreated samples (Ex vivo)


title: Hippocampus wild type 2

description: Wild type hippocampus region

tag: Wild type, whole hippocampus


title: cDC-2_U74A

source_name: Mouse Spleen conventional dendritic cells (cDC)

tag: BALB/c, female, 6-8 weeks old, spleen, FACS-sorted cDC, untreated samples (Ex vivo)


title: Hippocampus VC mice 5 weeks off dox

description: VP16-CREB hippocampus region

tag: 5 week removal of Doxycycline 40 mg/kg, VP16-CREB mice, whole hippocampus


title: IKDC-1_U74A

source_name: mouse spleen interferon producing killer-dendritic cells (IKDCs)

tag: BALB/c, female, 6-8 weeks old, spleen, FACS-sorted IKDC, untreated samples (Ex vivo)


title: cDC-2_U74C


title: Plasmacytoid DC1_U74C

source_name: Mouse Spleen Plasmacytoid DC (PDC)

tag: BALB/c, female, 6-8 weeks old, spleen, FACS-sorted PDC, untreated samples (Ex vivo)


title: IKDC-2_U74A

source_name: Mouse Spleen interferon producing killer-dendritic cells (IKDCs)

tag: BALB/c, female, 6-8 weeks old, spleen, FACS-sorted IKDC, untreated sample (Ex vivo)


title: cDC-1_U74A


title: Microdissected CA1 from wild type mice

description: Wild type hippocampus CA1 region

tag: Wild type, hippocampus CA1 region


title: Hippocampus wild type 1

description: Wild type hippocampus region


title: Plasmacytoid DC2_U74C


title: cDC-1_U74C


title: IKDC-2_U74C


title: Hippocampus VC mice on Dox

description: VP16-CREB hippocampus region

tag: Doxycycline 40 mg/kg, VP16-CREB mice, whole hippocampus


title: Plasmacytoid DC2_U74B


title: IKDC-1_U74C


title: IKDC-1_U74B


title: Plasmacytoid DC1_U74B


title: cDC-1_U74B


title: TaY-type2

city: Tokyo

first: Junko

middle: H

last: Ohyashiki

address: 6-7-1, Nishishinjuku, Shinjuku-ku

department: Intractable Immune System Diseases Research Center

country: Japan

institute: Tokyo Medical University

description:
Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA), then total mRNA was purified using Oligotex™-dT30<Super> mRNA Purification Kit (Takara, Japan). 1 microgram of mRNA were reverse transcribed into cDNA and labeled by cyanine 5 (Cy5)-conjugated deoxyuracil triphosphate (dUTP) or cyanine 3 (Cy3)-conjugated dUTP (Amerrsham Biosciences, Piscataway, NJ, USA) using LabelStar Array kit (Qiagen) , according to the supplier’s instruction.The normal lymphocyte mRNA obtained from human spleen (Ambion, Austin, TX, USA) was utilized as a reference in order to compare gene expression patterns of cells in both T and NK lineage. Hybridization was carried out automatically using GeneTAC Hybstation (Genomic Solutions, US) according to the supplier’s instruction. The conditions of hybridization were 55oC for 2 hours, 50oC for 2 hours, and 46oC for 2 hours, then 42oC for 12 hours. The hybridized signals were scanned by GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA, USA) as raw data. The scanned data was normalized, verified, and analyzed using the Genomic Profiler software (Mitsui Industrial Knowledge, Inc, Tokyo, Japan:http://bio.mki.co.jp/en/product/index.html).
Keywords = cancer array
Keywords = EB negative cell line

source_name: TaY cells (T cell leukemia cell line, EBV negative)

source_name: normal lymphocytes (reference)


title: Jutkat-type2

source_name: Jurkat (T cell leukemia cell line, EBV negative)


title: Molt-4-type2

source_name: Molt-4 (T cell leukemia cell line, EBV negative)


title: IKDC-2_U74B


title: Hippocampus VC mice 1 week off dox 2

description: VP16-CREB hippocampus region


title: SNK-10-type2

description:
Keywords = Chronic active EBV infection
Keywords = cancer array

hyb_protocol: Hybridization was carried out automatically using GeneTAC Hybstation (Genomic Solutions, AnnArbor, MI, US) according to the supplier’s instruction. The conditions of hybridization were 55oC for 2 hrs, 50oC for 2 hrs, and 46oC for 2 hrs, then 42oC for 12 hrs.

scan_protocol: The hybridized signals were scanned by GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA, USA) as raw data.

data_processing: The scanned data were normalized, verified, and analyzed using the Genomic Profiler software (Mitsui Industrial Knowledge Inc., Tokyo, Japan). First, the value was adjusted by subtraction of background fluorescence of an equivalent area. Analysis was done by taking the median signal of the probe value for each transcript set, and the 75% rank for the total hybridization was calculated. Microarray data obtained from three independent experiments were then verified in a single file. The normalized log data of fluorescence ratio (Cy5 to Cy3), which was quantified for each gene to reflect the relative abundance of gene in each experimental sample compared with reference sample, were deposited to GEO.

source_name: SNK-10 cells (NK cell line derived from patient with chronic active EBV infection)

label_protocol: 1 microgramof mRNA were reverse transcribed into cDNA and labeled by cyanine 5 (Cy5)-conjugated deoxyuracil triphosphate (dUTP) (Amersham Biosciences, Piscataway, NJ, USA) using a LabelStar Array kit (Qiagen), according to the supplier’s instruction.

growth_protocol: SNK-1 cells were maintained in RPMI1640 with 10% heat-inactivated human serum and 700 U/ml of IL-2.

extract_protocol: Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA). For microarray analysis, mRNA was purified using an Oligotex™-dT30<Super> mRNA Purification Kit (Takara, Shiga, Japan).

tag: SNK-1 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.

source_name: normal lymphocyte (reference)

label_protocol: LabelStar Array kit (Qiagen)

tag: Normal human lymphocyte mRNA was purchased from Ambion (Austin, TX, USA)


title: SNT-8-type2

description:
Keywords = nasal T cell lymphoma
Keywords = EBV
Keywords = cancer array

source_name: SNT-8 cells (nasal T cell lymphoma cell line)

growth_protocol: SNt-8 cells were maintained in RPMI1640 with 10% heat-inactivated human serum and 700 U/ml of IL-2.

tag: SNT-8 cells (nasal NK cell lymphoma cell line

value: EBV positive). SNT-8 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.

source_name: normal human lymphocytes

label_protocol: Normal human lymphocyte mRNA was purchased from Ambion (Austin, TX, USA)


title: cDC-2_U74B


title: SNK-6-type2

description:
Keywords = nasal NK cell lymphoma
Keywords = EBV
Keywords = cancer array

source_name: SNK-6 cells (nasal NK cell lymphoma cell line)

growth_protocol: SNK-6 cells were maintained in RPMI1640 with 10% heat-inactivated human serum and 700 U/ml of IL-2.

value: EBV positive). SNK-6 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.

label_protocol: a LabelStar Array kit (Qiagen)


title: SNT-16-type2

source_name: SNT-16 cells (T cell line established from a patient with chronic active EBV infection)

growth_protocol: Cells were maintained in RPMI1640 with 10% heat-inactivated human serum and 700 U/ml of IL-2.

extract_protocol: Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA). For microarray analysis, mRNA was purified using an Oligotex™-dT30<Super> mRNA Purification Kit (Takara, Shiga, Japan)

tag: SNT-16 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.


title: SNT-13-type2

source_name: SNT-13 cells (Tcell line established from a patient with chronic active EBV infection)

growth_protocol: SNT-13 cells were maintained in RPMI1640 with 10% heat-inactivated human serum and 700 U/ml of IL-2.

tag: SNT-13 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.


title: SNK-10-type1

description:
Keywords = chronic active EBV infection
Keywords = inflammatory response array

source_name: SNK-10 (NK cell line established from a patient with chronic active EBV infection


title: SNT-13-type1

source_name: SNT-13 (T cell line established from a patient with chronic active EBV infection.


title: SNK-1-type2

source_name: SNK-1 cells (nasal NK cell lymphoma cell line)

value: EBV positive). SNK-1 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.


title: SNT-16-type1

source_name: SNT-16 (T cell line derived from a patient with chronic active EBV infection)


title: Jurkat -type1

description:
Keywords = T cell line
Keywords = inflammatory response array

source_name: Jurkat (T cell leukemia cell line)

growth_protocol: Cells were maintained in RPMI1640 with 10% heat-inactivated FBS.

tag: EBV negative


title: SNK-1-type1

description:
Keywords = nasal NK cell lymphoma
Keywords = EBV
Keywords = inflammatory response array

hyb_protocol: Hybridization was carried out automatically using GeneTAC Hybstation (Genomic Solutions, AnnArbor, MI, US) according to the supplier’s instruction.  The conditions of hybridization were 55oC for 2 hrs, 50oC for 2 hrs, and 46oC for 2 hrs, then 42oC for 12 hrs.

source_name: SNK-1 cells (nasal NK cell lymphoma cell line: EBV positive)

value: EBV positive).  SNK-1 cells had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patient.


title: Molt4-type1

source_name: Molt4 (T cell leukemia cell line)


title: SNK-6-type1

description:
Keywords = nasal Nk cell lymphoma
Keywords = EBV
Keywords = inflammatory responese array

source_name: SNK-6 cells (nasal NK lymphoma cell line: EBV positive)

tag: SNK-6 cells (nasal NK cell lymphoma cell line

source_name: normal human lymphocyte


title: Eosinophils_control_rep2

city: Darlinghurst

middle: Reay

last: Mackay

state: NSW

address: 384 Victoria St

country: Australia

institute: Garvan Institute for Medical Research

description: 97% pure population of human peripheral blood eosinophils purified by Percoll and magnetic cell sorting (MACS) using anti-CD16, unstimulated

hyb_protocol: Fifteen micrograms of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer (40mM Tris-acetate (pH 8.1), 125mM KOAc, 30mM MgOAc). Hybridization cocktails were then made by adding fragmented cRNA, control cRNAs, grid alignment oligonucleotides and blocking reagents. These mixtures were hybridized for 16 hours to individual U133A and B genechips (Affymetrix, Santa Clara, CA) at 45 °C. Washing and staining of the hybridized arrays were performed using an Affymetrix Fluidics Station.

data_processing: GCOS

label: PE

source_name: Peripheral blood

label_protocol: cRNA was transcribed from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Farmingdale, NY).

extract_protocol:
Trizol was used for total RNA extraction.
cRNA was prepared according to methods for small RNA samples (Baugh et al, Nucleic acids research 29:E29, 2001). 500-1000 ng of RNA was reverse transcribed to cDNA using a poly(T) primer containing a T7-(dT)24 (Geneworks, Australia).

treatment_protocol:
Fresh blood (200 ml) was collected with EDTA and used immediately. Red blood cells were sedimented from whole blood in 0.6% dextran T500 (Pharmacia) for 30 minutes, at room temperature. Once red blood cells had sedimented to the bottom, the upper leukocyte-rich phase was carefully transferred to a new tube by pipetting. The leukocyte-rich phase was diluted 1:2 with wash buffer before overlaying onto a 70% and 80% discontinuous isotonic Percoll gradient.

Cells were centrifuged through the Percoll gradient at 1300 xg for 30 minutes, at room temperature. To avoid disturbing the gradient, brakes were turned off. After centrifugation, the granulocyte population, consisting mostly of neutrophils and eosinophils, were removed from the 70/80% interface. To minimise eosinophil activation, the cells were washed twice in cold wash buffer and centrifuged at 4°C. For the first wash, the cells were centrifuged at 680 xg, for 7 minutes, to remove Percoll. The supernatant was discarded and the pellet resuspended again in 50 ml of wash buffer before centrifugation at 470 xg, for 5 minutes. To remove the low numbers of contaminating red blood cells, the cell pellet was resuspended in 2 ml of 1x red blood cell lysis buffer and centrifuged immediately. After washing once in 2 ml of 1x lysis kit wash buffer, the cell pellet was resuspended in wash buffer and the cells were counted on a haemocytometer.

Neutrophils were depleted from the granulocyte population using anti-CD16 magnetic beads, according to the manufacturer's protocol (Miltenyi BioTec, Bergisch Gladbach, Germany). After the granulocytes were counted, they were resuspended in MACS buffer (50 μl/5 × 107 total cells). Anti-CD16 magnetics beads (Miltenyi Biotec) were also added (50 μl/5 × 107 total cells). Cells were incubated with anti-CD16 magnetic beads at 6–12°C for 30–40 minutes. Depletion columns and flow resistors were used, according to the manufacturer's protocol (Miltenyi Biotec). The columns were assembled onto a Varios MACS magnetic apparatus (Miltenyi Biotec) and washed with MACS buffer. After incubation, the cell suspension was diluted with MACS buffer (5 × 107 total cells/ml) before applying to the column. CD16 positive cells, including neutrophils, were retained in the column by the magnetic force. Eosinophils were collected into a chilled 15 ml tube on ice. Eosinophils were stained with trypan blue and counted on a haemocytometer. Cell viability according to trypan blue exclusion was >97%. To determine the purity of the sample, the cells were cytocentrifuged onto a slide and stained with Wright's modified Giemsa stain (Sigma Diagnostics), according to the manufacturer's instructions. At least 400 cells were counted on each slide. Only samples with >97% eosinophils were used in further experiments. The main contaminating cells were neutrophils.

tag: Human peripheral blood purified eosinophils (unstimulated)


title: Wild-type TG-macrophages_LPS 6h

status: Public on Jan 10 2006

description: Wild-type thioglycollate-elicited macrophages stimulated with LPS for 6 hours

source_name: peritoneal lavage

extract_protocol: Trizol was used for total RNA extraction. cRNA was prepared according to methods for small RNA samples (Baugh et al, Nucleic acids research 29:E29, 2001). 500-1000 ng of RNA was reverse transcribed to cDNA using a poly(T) primer containing a T7-(dT)24 (Geneworks, Australia).

treatment_protocol: Pac1+/+ mice were injected intraperitoneally with 2 mL of 3% thioglycollate (Sigma) 4 days prior to sacrifice.  Peritoneal macrophages were collected by lavaging the peritoneal cavity with 10 ml of sterile RPMI 1640 medium.  Macrophages were purified by adherence to tissue culture plates for 2 h.  The macrophage population was approximately 92-95% mac-1 positive as assessed by PE-labelled mac-1 antibodies (BD Pharmingen).  Morphology was assessed by cytospin and staining with giemsa.  Peritoneal macrophages at 2 × 10^6 cells/mL were treated with 100 ng/ml of LPS (Sigma, St Louis Lot # 072K4096) for 6 hours.

tag: Thioglycollate-elicited (72 h) peritoneal macrophages from wild-type mice stimulated with 100 ng/ml of LPS for 6 hours


title: 23a_HT4

city: University Park

first: Jordan

middle: D

last: Irvin

state: PA

address: 452 N Frear

department: Biochemistry

institute: Penn State University

description: no additional information needed

hyb_protocol: see above

data_processing: MeanFRG- MedianBCK>S.D. BCK, <26% saturation.  Ratio of Test/Ref.

source_name: strain yjdi363

growth_protocol: Cells were grown in CSM-LEU + 3% raffinose to OD600 of ~0.7.  Galactose was added to 2% and cultures were incubated for 30 minutes at 25ûC.  Cultures were then rapidly shifted to 37ûC by adding warm media and placing in a 37ûC shaker incubator for 45 minutes before harvest at 4ûC.

extract_protocol: Holstege et al.  1998.  Cell.  95:717-28

treatment_protocol: Galactose induction of TAF1 mutants and heat shock (37C), see below.

tag: strain yjdi363 GAL1-TAF1 WT + taf1 ts2

source_name: strain yjdi359

label_protocol: [Chitikila et al.  2002. Mol Cell. 10:871-82

growth_protocol: see above

extract_protocol: see above

treatment_protocol: see above

tag: strain yjdi359 GAL1-taf1 FHT-HT4 + taf1 ts2


title: 24a_null

description: no additional information needed

scan_protocol: Chitikila et al.  2002. Mol Cell. 10:871-82

source_name: strain yjdi381

tag: strain yjdi381 GAL1-taf1::KanMX6 + taf1 ts2


title: 4a_homotypic

description: no additional information needed

label_protocol: Chitikila et al.  2002. Mol Cell. 10:871-82


title: 23b_PB1

description: no additional information needed

source_name: strain yjdi361

tag: strain yjdi361 GAL1-taf1 FHT-PB1 + taf1 ts2


title: 27a600660_homotypic

description: no additional information needed


title: 22a640600_DT1

description: no additional information needed

source_name: strain yjdi355

tag: strain yjdi355 GAL1-taf1 FHT-DT1 + taf1 ts2


title: 4b_FHT-WT1

description: no additional information needed

source_name: strain yjdi353

tag: strain yjdi353 GAL1-taf1 FHT-WT1 + taf1 ts2


title: 0h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 6

type: mixed

city: Montreal

first: Sébastien

middle: P

last: Faucher

state: QUEBEC

address: C.P. 6128 Succ. Centre-Ville

country: Canada

institute: University of Montreal

description: This sample was used to analyse the expression profiles of Salmonella typhi during infection of human macrophages (THP-1)

hyb_protocol: Samples we hybridized as previously described (Porwolik, S. 2003.Nucleic Acids Res 31:1869-76)

scan_protocol: Slide was scanned with Scan Array Lite (Perkin-Elmer) using Scan Array Express 2.1 software.

data_processing: After backgroud correction and total intensity normalization, ratio to reference were calculated and log base 2 transformed.

organism: Salmonella enterica subsp. enterica serovar Typhi

source_name: 3XSCOTS cDNA obtained 0h post-infection

label_protocol: 1.5 µg of cDNA was labeled with Cy5-dCTP (GE Healthcare) by random priming with Klenow (New England Biolabs) as previously described (Porwollik, S. 2001. Mutat Res 483:1-11.).

extract_protocol: TRIzol reagent (Gibco BRL) was used according to the manufacturer instruction. RNA was treated with DNase (Ambion). RNA sample was converted to cDNA in 5 independent reverse-transcription reactions as described previously (Graham, J. 1999. PNAS 96:11554-559). Bacterial transcript were purified from host transcript by using 3 round of the SCOTS procedure (Daigle, F. 2002. Methods Enzymol. 358:108-122).

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, cells were washed with PBS and recovered in 5 ml PBS. After centrifuation, the pellet was lysed with 2 ml TRIzol (Invitrogen).

tag: Salmonella typhi infection of THP-1 cells, recovered 0 h post-infection

source_name: Reference genomic DNA

label_protocol: 1.5 µg of genomic DNA was labeled with Cy3-dCTP (GE Healthcare) by random priming with Klenow (New England Biolabs) as previously described (Porwollik, S. 2001. Mutat Res 483:1-11.).

growth_protocol: Salmonella typhi from overnight culture in LB broth.

extract_protocol: Genomic DNA was extracted by standard phenol-chloroform method (Sambrook, J, Molecular Cloning, 3rd Edition)

tag: Genomic DNA from Salmonella typhi ISP1820


title: 22b640610_TF1

description: no additional information needed

source_name: strain yjdi357

tag: strain yjdi357 GAL1-taf1 FHT-TF1 + taf1 ts2


title: 28a630650_null

description: no additional information needed


title: 0h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 5


title: Supernatant of infection treated with 3 rounds of SCOTS, replicate 5

source_name: 3XSCOTS cDNA obtained from infection supernatant

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, the supernatant was removed and centrifuge to recover the bacteria. The pellet was lysed with 2 ml of TRIzol (Gibco BRL)

tag: Salmonella typhi infection of THP-1 cells, recovered from the culture medium supernatant


title: 0h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 4


title: Pac-1-/- TG-macrophages_LPS 6h

description: Pac-1-/- thioglycollate-elicited macrophages stimulated with LPS for 6 hours

treatment_protocol: Pac1–/– mice were injected intraperitoneally with 2 mL of 3% thioglycollate (Sigma) 4 days prior to sacrifice.  Peritoneal macrophages were collected by lavaging the peritoneal cavity with 10 ml of sterile RPMI 1640 medium.  Macrophages were purified by adherence to tissue culture plates for 2 h.  The macrophage population was approximately 92-95% mac-1 positive as assessed by PE-labelled mac-1 antibodies (BD Pharmingen).  Morphology was assessed by cytospin and staining with giemsa.  Peritoneal macrophages at 2 × 10^6 cells/mL were treated with 100 ng/ml of LPS (Sigma, St Louis Lot # 072K4096) for 6 hours.

tag: Thioglycollate-elicited (72 h) peritoneal macrophages from PAC-1 deficient mice stimulated with 100 ng/ml of LPS for 6 hours


title: Supernatant of infection treated with 3 rounds of SCOTS, replicate 4


title: SNT-8-type1

description:
Keywords = nasal NK/T lymphoma
Keywords = EBV
Keywords = inflammatory resonese array

source_name: SNT-8 (nasal T cell lymphoma cell line: EBV positive)

tag: SNT-8 cells (nasal NK cell lymphoma cell line

source_name: normal human lymphocytes

tag: Normal human lymphocyte mRNA was purchased from Ambion (Austin, TX, USA)


title: 25b620610_DT1

description: no additional information needed

tag: strain yjdi355 GAL1-FHT taf1 DT1 + taf1 ts2


title: 27b630620_PB1

description: no additional information needed

tag: strain yjdi361 GAL1-FHT taf1 PB1 + taf1 ts2


title: 26a610610_TF1

description: no additional information needed

tag: strain yjdi357 GAL1-FHT taf1 TF1 + taf1 ts2


title: 26b620600_HT4

description: no additional information needed

tag: strain yjdi359 GAL1-FHT taf1 HT4 + taf1 ts2


title: Comparative Genomic Hybridization between strain ISP1820 and CT18, replcate 3

description: This sample was used to identify genes absent in ISP1820 compared to CT18

source_name: Salmonella typhi ISP1820 genomic DNA

growth_protocol: Salmonella typhi ISP1820 from overnight culture in LB broth.

source_name: Salmonella typhi CT18 genomic DNA

tag: Genomic DNA from Salmonella typhi CT18


title: 8h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 4

source_name: 3XSCOTS cDNA obtained 8h post-infection

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, cells were washed with PBS and fresh RPMI containing 100 ug ml-1 was added to the wells. After 2 hours, cells were washed with PBS and fresh RPMI containing 12 ug ml-1 was added to the wells. After 8 hours, cells were washed with PBS and recovered in 5 ml PBS. After centrifuation, the pellet was lysed with 2 ml TRIzol (Invitrogen).

tag: Salmonella typhi infection of THP-1 cells, recovered 8 h post-infection


title: Comparative Genomic Hybridization between strain ISP1820 and CT18, replicate 1


title: 0h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 1


title: 8h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 6


title: 24h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 2

source_name: 3XSCOTS cDNA obtained 24h post-infection

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, cells were washed with PBS and fresh RPMI containing 100 ug ml-1 was added to the wells. After 2 hours, cells were washed with PBS and fresh RPMI containing 12 ug ml-1 was added to the wells. After 24 hours, cells were washed with PBS and recovered in 5 ml PBS. After centrifuation, the pellet was lysed with 2 ml TRIzol (Invitrogen).

tag: Salmonella typhi infection of THP-1 cells, recovered 24 h post-infection


title: 25a660650_FHT-WT1

description: no additional information needed

tag: strain yjdi353 GAL1-FHT taf1 WT1 + taf1 ts2

tag: strain yjdi363 GAL1-TAF1 WT + taf1 ts2


title: 0h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 2


title: 8h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 3


title: 24h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 4


title: 24h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 5


title: 0h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 3


title: 24h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 3


title: 8h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 1


title: 8h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 2


title: Supernatant of infection treated with 3 rounds of SCOTS, replicate 2


title: Supernatant of infection treated with 3 rounds of SCOTS, replicate 3


title: 24h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 1


title: 8h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 5


title: 24h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 6


title: Th2 cells_unstimulated_rep1

description: Human neonatal blood-derived type II helper T cells, ~95% pure.

source_name: Cord blood

treatment_protocol: Human neonatal leukocytes were isolated from heparinized cord blood by Ficoll density gradient centrifugation. CD4+ T cells were obtained using a CD4 negative selection kit (Miltenyi Biotec). Polarized Th2 cells were generated as previously described (Sornasse et al., 1996) by stimulation with immobilized anti-CD3 (clone TR66), soluble anti-CD28 (BD Pharmingen) and IL-2. Th2 cultures also received IL-4 (BD Pharmingen) and neutralizing anti-INF-g (BD Pharmingen). The cells were cultured for 3-4 days, harvested and restimulated under the same conditions for another 3-4 days. Cells were then harvested for RNA extraction.

tag: Human Th2 cells


title: Female Placebo-Treated Submandibular Gland Sample C

first: Nathaniel

last: Treister

address: 20 Staniford Street

institute: Schepens Eye Research Institute

description: Young adult female BALB/c mice (n = 30), ovariectomized at age 8 weeks, were purchased from Taconic Laboratories (Germantown, NY) and maintained in constant temperature rooms with fixed light/dark periods of 12 hours.  Animals were allowed to recover from surgery for 10 days, then anesthetized with ketamine and xylazine, and subcutaneous implants of placebo (n = 15; cholesterol, methylcellulose, lactose)- or testosterone (n = 15; 10 mg)-containing pellets were inserted in the subscapular region.  Hormone pellets were obtained from Innovative Research of America (Sarasota, FL) and were designed to maintain male physiologic amounts of testosterone over a two week period.  Following two weeks of hormone exposure, the mice were sacrificed in a CO2 chamber, and the lacrimal, meibomian, and submandibular glands were removed and pooled (5 mice per group) into three groups by treatment (n = 6 groups).  Glands were immediately frozen and stored in liquid nitrogen until RNA isolation.

data_processing: The 10,000 spot intensities on the scanned microarray image were normalized to a median value of 1 and data were exported for analysis.

label: CodeLink Expression Assay Reagent Kit

source_name: Balb/c Placebo-Treated Submandibular Gland

tag: Series includes pooled (n = 5 mice per biological replicate) samples from lacrimal, meibomian, and submandibular glands of female palcebo- and testosterone-treated BALB/c mice. All mice were ovariectomized at 8 weeks and allowed to recover from surgery for 10 days prior to hormone implanatation. All experiments were run in triplicate (pooled biological replicates) on CodeLink Mouse Uniset I Microarrays.


title: Central memory T cells_unstimulated_rep2

description: Human peripheral blood CD4+CD45RO+CCR7+ central memory T cells, unstimulated, >95% pure.

source_name: Peripheral blood

treatment_protocol: Fresh blood was collected in EDTA. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers by centrifugation over Ficoll Paque (Amersham Biosciences, Uppsala, Sweden) and washed. CD4+CD45RO+CCR7+ cells were sorted from PBMC using a FACSVantage SE DiVa® (BD Biosciences, San Jose, CA).


title: Female Placebo-Treated Submandibular Gland Sample A

description: Young adult female BALB/c mice (n = 30), ovariectomized at age 8 weeks, were purchased from Taconic Laboratories (Germantown, NY) and maintained in constant temperature rooms with fixed light/dark periods of 12 hours. Animals were allowed to recover from surgery for 10 days, then anesthetized with ketamine and xylazine, and subcutaneous implants of placebo (n = 15; cholesterol, methylcellulose, lactose)- or testosterone (n = 15; 10 mg)-containing pellets were inserted in the subscapular region. Hormone pellets were obtained from Innovative Research of America (Sarasota, FL) and were designed to maintain male physiologic amounts of testosterone over a two week period. Following two weeks of hormone exposure, the mice were sacrificed in a CO2 chamber, and the lacrimal, meibomian, and submandibular glands were removed and pooled (5 mice per group) into three groups by treatment (n = 6 groups). Glands were immediately frozen and stored in liquid nitrogen until RNA isolation.


title: Female Placebo-Treated Submandibular Gland Sample B


title: Female Testosterone-Treated Submandibular Gland Sample C

source_name: Balb/c Testosterone-Treated Submandibular Gland


title: 2h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 5

description: This sample was used to analyse the effect of SCOTS on transcript population and to analyse the expression profiles of Salmonella typhi during infection of human macrophages (THP-1)

source_name: 3XSCOTS cDNA obtained 2h post-infection

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, cells were washed with PBS and fresh RPMI containing 100 ug ml-1 was added to the wells. After 2 hours, cells were washed with PBS and recovered in 5 ml PBS. After centrifuation, the pellet was lysed with 2 ml TRIzol (Invitrogen).

tag: Salmonella typhi infection of THP-1 cells, recovered 2 h post-infection

tag: Genomic DNA from Salmonella typhi ISP1820


title: Th1 cells_unstimulated_rep1

description: Human neonatal blood-derived type I helper T cells, ~95% pure.

treatment_protocol: Human neonatal leukocytes were isolated from heparinized cord blood by Ficoll density gradient centrifugation. CD4+ T cells were obtained using a CD4 negative selection kit (Miltenyi Biotec). Polarized Th1 cells were generated as previously described (Sornasse et al., 1996) by stimulation with immobilized anti-CD3 (clone TR66), soluble anti-CD28 (BD Pharmingen) and IL-2. Th1 cultures also received IL-12 (BD Pharmingen) and neutralizing anti-IL-4 (BD Pharmingen). The cells were cultured for 3-4 days, harvested and restimulated under the same conditions for another 3-4 days. Cells were then harvested for RNA extraction.

tag: Human Th1 cells


title: Female Testosterone-Treated Submandibular Gland Sample A


title: Female Testosterone-Treated Submandibular Gland Sample B


title: 2h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 6


title: Th1 cells_unstimulated_rep2


title: Female Testosterone-Treated Meibomian Gland Sample C

source_name: Balb/c Testosterone-Treated Meibomian Gland


title: 2h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 2


title: Comparative Genomic Hybridization between strain ISP1820 and CT18, replcate 2


title: Female Testosterone-Treated Meibomian Gland Sample A


title: Basophils_unstimulated_rep2

description: Human CCR3+, low side scatter peripheral blood basophils, unstimulated, basophils were >96% pure.

treatment_protocol: Fresh blood (200 ml) was collected in EDTA. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers by centrifugation over Ficoll Paque (Amersham Biosciences, Uppsala, Sweden) and washed. CCR3+ basophils were sorted from PBMC using a FACSVantage SE DiVa® (BD Biosciences, San Jose, CA) after CD3+ and CD14+ cells were depleted by MACS (Miltenyi Biotec). Doublets and aggregated cells were excluded to minimise contaminating cells.

tag: Human peripheral blood CCR3+ basophils, unstimulated


title: Female Testosterone-Treated Meibomian Gland Sample B


title: Central memory T cells_unstimulated_rep1

tag: Human peripheral blood CD4+CD45RO+CCR7+ central memory T cells


title: 2h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 1


title: 2h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 3


title: 2h post-infection of THP-1 treated with 3 rounds of SCOTS, replicate 4


title: Female Placebo-Treated Meibomian Gland Sample C

source_name: Balb/c Placebo-Treated Meibomian Gland


title: Th2 cells_unstimulated_rep2


title: Female Placebo-Treated Meibomian Gland Sample B


title: NK cells_unstimulated_rep2

description: Human peripheral blood CD16+CD56+ NK cells, unstimulated, >96% pure.

treatment_protocol: Fresh blood was collected in EDTA. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers by centrifugation over Ficoll Paque (Amersham Biosciences, Uppsala, Sweden) and washed. CD16+CD56+ cells were sorted from PBMC using a FACSVantage SE DiVa® (BD Biosciences, San Jose, CA). Doublets and aggregated cells were excluded to minimise contaminating cells.

tag: Human CD16+CD56+, peripheral blood NK cells, unstimulated


title: Female Placebo-Treated Meibomian Gland Sample A


title: 2h post-infection of THP-1 treated with 2 rounds of SCOTS, replicate 5

description: This sample was used to analyse the effect of SCOTS on transcript population.

source_name: 2XSCOTS cDNA obtained 2h post-infection

extract_protocol: TRIzol reagent (Gibco BRL) was used according to the manufacturer instruction. RNA was treated with DNase (Ambion). RNA sample was converted to cDNA in 5 independent reverse-transcription reactions as described previously (Graham, J. 1999. PNAS 96:11554-559). Bacterial transcript were purified from host transcript by using 2 round of the SCOTS procedure (Daigle, F. 2002. Methods Enzymol. 358:108-122).


title: Effector memory T cells_unstimulated_rep2

description: Human peripheral blood CD4+CD45RO+CCR7- effector memory T cells, unstimulated, >95% pure.

treatment_protocol: Fresh blood was collected in EDTA. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers by centrifugation over Ficoll Paque (Amersham Biosciences, Uppsala, Sweden) and washed. CD4+CD45RO+CCR7- cells were sorted from PBMC using a FACSVantage SE DiVa® (BD Biosciences, San Jose, CA).


title: Female Testosterone-Treated Lacrimal Gland Sample B

source_name: Balb/c Testosterone-Treated Lacrimal Gland


title: Female Testosterone-Treated Lacrimal Gland Sample A


title: NK cells_unstimulated_rep1


title: Female Placebo-Treated Lacrimal Gland Sample C

source_name: Balb/c Placebo-Treated Lacrimal Gland


title: Effector memory T cells_unstimulated_rep1

treatment_protocol: Fresh blood (200 ml) was collected in EDTA. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers by centrifugation over Ficoll Paque (Amersham Biosciences, Uppsala, Sweden) and washed. CD4+CD45RO+CCR7- cells were sorted from PBMC using a FACSVantage SE DiVa® (BD Biosciences, San Jose, CA).

tag: Human peripheral blood CD4+CD45RO+CCR7- effector memory T cells


title: 2h post-infection of THP-1 treated with 2 rounds of SCOTS, replicate 6

source_name: Reference genomic DNA


title: Microdissected CA1 region from VC mice 3 weeks off dox 2

description: VP16-CREB hippocampus CA-1 region

source_name: hippocampus CA1 region

tag: 3 weeks removal of Doxycycline 40 mg/kg, VP16-CREB mice, hippocampus CA1 region


title: Female Placebo-Treated Lacrimal Gland Sample A

tag: Series includes pooled (n = 5 mice per biological replicate) samples from lacrimal, meibomian, and submandibular glands of female palcebo- and testosterone-treated BALB/c mice. All mice were ovariectomized at 8 weeks and allowed to recover from surgery for 10 days prior to hormone implanatation.  All experiments were run in triplicate (pooled biological replicates) on CodeLink Mouse Uniset I Microarrays.


title: Female Placebo-Treated Lacrimal Gland Sample B


title: Pac1-/- BMMCs_IgE-DNP 2h_rep2

description: Pac1-/- bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours where cells were sensitized with IgE anti-DNP for 18 hours and receptors cross-linked with DNP-HSA antigen for 2 hours.

source_name: Bone marrow

treatment_protocol: Bone marrow was isolated from femurs and tibias of 8-12 week-old Pac1-/- mice.  Cells were grown at 4 × 10^5 cells/ml in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 μg each of penicillin and streptomycin per ml, 2 mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES, pH 7.5 and 5 ng/ml of recombinant murine IL-3 (Peprotech) for 4-6 weeks at 37 oC in 5% CO2.  By 4 - 6 weeks in culture, greater than 98% of cells were c-kit and FcεRI positive as assessed by fluorescein isothiocyanate (FITC)-labelled anti-c-kit (BD, Pharmingen) and anti-DNP IgE (Sigma) antibodies, respectively.  Cell morphology was also assessed by cytospin and staining with giemsa and toluidine blue.  To stimulate, cells were sensitised with anti-DNP IgE mAb (Sigma, 100 ng/ml) for 18 hours, washed twice to remove unbound IgE and stimulated with 20 ng/ml of DNP-HSA (Sigma) for 2 hours.

tag: Pac1-/- bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours.


title: Neutrophils_LPS 1h_rep1

description: Human CD16+, peripheral blood neutrophils, LPS-stimulated for 1 h, neutrophils were >96% pure.

treatment_protocol:
Fresh blood (50 ml) was collected in EDTA and used immediately. Whole blood was centrifuged at 300 xg for 20 minutes, at room temperature. The upper plasma layer was removed from the cell pellet and centrifuged at 1800 xg for 15 minutes, at room temperature, to remove platelets. An equal volume of 1x PBS was used to replace the volume of plasma removed before red blood cells were sedimented in 0.6% dextran T500. After 30 to 45 minutes, the upper leukocyte-rich phase was carefully transferred to a new tube and centrifuged at 170 xg for 5 minutes, to further remove platelets. The cell pellet was then resuspended in platelet-poor plasma before carefully overlaying onto a 70% and 80% discontinuous isotonic Percoll gradient.

The cells were centrifuged through the discontinuous Percoll gradient at 900 xg for 30 minutes, at room temperature. To avoid disturbing the gradient, the brakes were turned off. After centrifugation, the granulocyte population, consisting mostly of neutrophils and eosinophils, were removed from the 70/80% interface. To minimise neutrophil apoptosis, the cells were washed three times in unchilled wash buffer and centrifuged at room temperature. For the first wash, the cells were centrifuged at 170 xg, for 10 minutes, to remove Percoll. The supernatant was discarded and the pellet resuspended again in 50 ml of wash buffer before centrifugation at 170 xg, for 5 minutes. The cells were then resuspended in wash buffer and counted on a haemocytometer. To determine the number of contaminating eosinophils, the cells were cytocentrifuged onto a slide and stained with Wright's modified giemsa stain, according to the manufacturer's protocol, and 300 cells were counted.

To avoid neutrophil apoptosis, MACS was performed at room temperature. Supernatant containing anti-CCR3 monoclonal antibodies (mAb) (10 mg/ml) was collected from hybridoma cultures. After washing, the granulocyte cell pellet was resuspended in anti-CCR3 mAb supernatant at a concentration of 4 μg per 106 CCR3 positive cells, determined empirically by counting the percentage of eosinophils in the granulocyte sample. The granulocytes were incubated in hybridoma supernatant for 10 minutes, at room temperature. To wash off excess mAb, the cells were washed twice in wash buffer by centrifuging at 300 xg for 5 minutes. The cells were then resuspended in 80 μL of wash buffer, and 20 μL of goat anti-mouse IgG microbeads (Miltenyi Biotec), per 10^7 total cells and incubated for 5 minutes at room temperature. After incubation, the cells were washed twice in wash buffer before they were put through a negative selection column (Miltenyi Biotec), according to the manufacturer's protocols.
The flow-through was collected and the number of neutrophils and cell viability was determined by counting trypan blue stained cells on a hemocytometer. Cell viability according to trypan blue was >98%. To determine the sample purity, the cells were cytocentrifuged onto slides and stained with Wright's modified giemsa stain, and 300 cells were counted. Only samples with >97 % neutrophils were used for microarray analysis. Neutrophils were stimulated for 1 h with 100 ng/ml LPS.

tag: Human CD16+, CCR3- peripheral blood neutrophils, LPS-stimulated for 1 h


title: Basophils_unstimulated_rep1


title: 2h post-infection of THP-1 treated with 2 rounds of SCOTS, replicate 2


title: 2h post-infection of THP-1 treated with 2 rounds of SCOTS, replicate 3


title: 2h post-infection of THP-1 treated with 2 rounds of SCOTS, replicate 4


title: Wild-type BMMCs_IgE-DNP 2h_rep2

description: Wild-type bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours where cells were sensitized with IgE anti-DNP for 18 hours and receptors cross-linked with DNP-HSA antigen for 2 hours.

treatment_protocol: Bone marrow was isolated from femurs and tibias of 8-12 week-old Pac1+/+ mice.  Cells were grown at 4 × 10^5 cells/ml in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 μg each of penicillin and streptomycin per ml, 2 mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES, pH 7.5 and 5 ng/ml of recombinant murine IL-3 (Peprotech) for 4-6 weeks at 37 oC in 5% CO2.  By 4 - 6 weeks in culture, greater than 98% of cells were c-kit and FcεRI positive as assessed by fluorescein isothiocyanate (FITC)-labelled anti-c-kit (BD, Pharmingen) and anti-DNP IgE (Sigma) antibodies, respectively.  Cell morphology was also assessed by cytospin and staining with giemsa and toluidine blue.  To stimulate, cells were sensitised with anti-DNP IgE mAb (Sigma, 100 ng/ml) for 18 hours, washed twice to remove unbound IgE and stimulated with 20 ng/ml of DNP-HSA (Sigma) for 2 hours.

tag: Wild-type bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours.


title: B cells_unstimulated_rep2

description: Human CD19+, peripheral blood B cells, unstimulated, cells were >96% pure.

treatment_protocol: Blood was collected in EDTA tubes. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers by centrifugation over Ficoll Paque (Amersham Biosciences, Uppsala, Sweden) and washed. CD19+ B cells cells were sorted from PBMC using a FACSVantage SE DiVa® (BD Biosciences, San Jose, CA). Doublets and aggregated cells were excluded to minimise contaminating cells.


title: Wild-type BMMCs_IgE-DNP 2h_rep1

description: Wild-type bone marrow-derived mast cells stimulated with Fc epsilon receptor crosslinking for 2 hours where cells were sensitized with IgE anti-DNP for 18 hours and receptors cross-linked with DNP-HSA antigen.

treatment_protocol: .Bone marrow was isolated from femurs and tibias of 8-12 week-old Pac1+/+ mice.  Cells were grown at 4 × 10^5 cells/ml in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 μg each of penicillin and streptomycin per ml, 2 mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES, pH 7.5 and 5 ng/ml of recombinant murine IL-3 (Peprotech) for 4-6 weeks at 37 oC in 5% CO2.  By 4 - 6 weeks in culture, greater than 98% of cells were c-kit and FcεRI positive as assessed by fluorescein isothiocyanate (FITC)-labelled anti-c-kit (BD, Pharmingen) and anti-DNP IgE (Sigma) antibodies, respectively.  Cell morphology was also assessed by cytospin and staining with giemsa and toluidine blue.  To stimulate, cells were sensitised with anti-DNP IgE mAb (Sigma, 100 ng/ml) for 18 hours, washed twice to remove unbound IgE and stimulated with 20 ng/ml of DNP-HSA (Sigma) for 2 hours.


title: 2h post-infection of THP-1 treated with 1 rounds of SCOTS, replicate 6

source_name: 1XSCOTS cDNA obtained 2h post-infection

extract_protocol: TRIzol reagent (Gibco BRL) was used according to the manufacturer instruction. RNA was treated with DNase (Ambion). RNA sample was converted to cDNA in 5 independent reverse-transcription reactions as described previously (Graham, J. 1999. PNAS 96:11554-559). Bacterial transcript were purified from host transcript by using 1 round of the SCOTS procedure (Daigle, F. 2002. Methods Enzymol. 358:108-122).


title: 2h post-infection of THP-1 treated with 2 rounds of SCOTS, replicate 1


title: Neutrophils_unstimulated_rep2

description: >97% pure population of CD16+, CD62L high, CCR3- peripheral blood neutrophils, unstimulated

label_protocol: cRNA was transcribed from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Farmingdale, NY)

treatment_protocol:
Fresh blood (50 ml) was collected in EDTA and used immediately. Whole blood was centrifuged at 300 xg for 20 minutes, at room temperature. The upper plasma layer was removed from the cell pellet and centrifuged at 1800 xg for 15 minutes, at room temperature, to remove platelets. An equal volume of 1x PBS was used to replace the volume of plasma removed before red blood cells were sedimented in 0.6% dextran T500. After 30 to 45 minutes, the upper leukocyte-rich phase was carefully transferred to a new tube and centrifuged at 170 xg for 5 minutes, to further remove platelets. The cell pellet was then resuspended in platelet-poor plasma before carefully overlaying onto a 70% and 80% discontinuous isotonic Percoll gradient.

The cells were centrifuged through the discontinuous Percoll gradient at 900 xg for 30 minutes, at room temperature. To avoid disturbing the gradient, the brakes were turned off. After centrifugation, the granulocyte population, consisting mostly of neutrophils and eosinophils, were removed from the 70/80% interface. To minimise neutrophil apoptosis, the cells were washed three times in unchilled wash buffer and centrifuged at room temperature. For the first wash, the cells were centrifuged at 170 xg, for 10 minutes, to remove Percoll. The supernatant was discarded and the pellet resuspended again in 50 ml of wash buffer before centrifugation at 170 xg, for 5 minutes. The cells were then resuspended in wash buffer and counted on a haemocytometer. To determine the number of contaminating eosinophils, the cells were cytocentrifuged onto a slide and stained with Wright's modified giemsa stain, according to the manufacturer's protocol, and 300 cells were counted.

To avoid neutrophil apoptosis, MACS was performed at room temperature. Supernatant containing anti-CCR3 monoclonal antibodies (mAb) (10 mg/ml) was collected from hybridoma cultures. After washing, the granulocyte cell pellet was resuspended in anti-CCR3 mAb supernatant at a concentration of 4 μg per 106 CCR3 positive cells, determined empirically by counting the percentage of eosinophils in the granulocyte sample. The granulocytes were incubated in hybridoma supernatant for 10 minutes, at room temperature. To wash off excess mAb, the cells were washed twice in wash buffer by centrifuging at 300 xg for 5 minutes. The cells were then resuspended in 80 μL of wash buffer, and 20 μL of goat anti-mouse IgG microbeads (Miltenyi Biotec), per 10^7 total cells and incubated for 5 minutes at room temperature. After incubation, the cells were washed twice in wash buffer before they were put through a negative selection column (Miltenyi Biotec), according to the manufacturer's protocols.
The flow-through was collected and the number of neutrophils and cell viability was determined by counting trypan blue stained cells on a hemocytometer. Cell viability according to trypan blue was >98%. To determine the sample purity, the cells were cytocentrifuged onto slides and stained with Wright's modified giemsa stain, and 300 cells were counted. Unactivated neutrophils (high CD62L) were also detected by FACS analysis using a phycoerythrin-conjugated anti-human CD62L antibody (Pharmingen). Only samples with >97 % neutrophils (high CD62L) were used for microarray analysis. The main contaminating cells were eosinophils. No monocytes were detected on cytospin slides.

tag: Human CD16+, CD62L high, CCR3- peripheral blood neutrophils, unstimulated


title: 2h post-infection of THP-1 treated with 1 rounds of SCOTS, replicate 5


title: 2h post-infection of THP-1 treated with 1 rounds of SCOTS, replicate 2


title: Macrophages_LPS 4h_rep2

description: Mature peripheral blood CD14+ monocyte-derived macrophages differentiated using GM-CSF over 11 days, LPS-stimulated (4 hours).

treatment_protocol: Macrophages were differentiated from isolated monocytes in the presence of GM-CSF. Briefly, peripheral blood was obtained from healthy volunteers and diluted 1:2 with PBS (GibcoBRL, New York, USA). Mononuclear leukocytes were isolated using a Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient and washed twice with PBS containing 2% FCS and 2mM EDTA. Monocytes were selected using anti-CD14 magnetic beads, according to the manufacturer’s protocol (Miltenyi BioTec, Bergisch Gladbach, Germany). The purified monocytes were resuspended at a cell concentration of 1x106 cells/ml in complete medium supplemented with 500 U/ml GM-CSF (BD PharMingen, Franklin Lakes, USA) and cultured for 11 days. Cultures were fed every 3 days by removing part of the supernatant and adding back fresh supplemented media. Adherent cells showed the typical morphology of macrophages. Macrophages were activated for 4 h with 100 ng/ml of LPS.

tag: Macrophages differentiated from isolated CD14+ monocytes in the presence of GM-CSF, LPS-stimulated for 4 hours.


title: 2h post-infection of THP-1 treated with 1 rounds of SCOTS, replicate 3


title: Macrophages_LPS 4h_rep1

extract_protocol:
Trizol was used for total RNA extraction. cRNA was prepared according to methods for small RNA samples (Baugh et al,
Nucleic acids research 29:E29, 2001). 500-1000 ng of RNA was reverse transcribed to cDNA using a poly(T) primer containing a T7-(dT)24 (Geneworks, Australia).

tag: Macrophages differentiated from isolated CD14+ monocytes in the presence of GM-CSF, LPS-stimulated for 4 hours


title: Pac1-/- BMMCs_IgE-DNP 2h_rep1

treatment_protocol: .Bone marrow was isolated from femurs and tibias of 8-12 week-old Pac1-/- mice.  Cells were grown at 4 × 10^5 cells/ml in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 μg each of penicillin and streptomycin per ml, 2 mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES, pH 7.5 and 5 ng/ml of recombinant murine IL-3 (Peprotech) for 4-6 weeks at 37 oC in 5% CO2.  By 4 - 6 weeks in culture, greater than 98% of cells were c-kit and FcεRI positive as assessed by fluorescein isothiocyanate (FITC)-labelled anti-c-kit (BD, Pharmingen) and anti-DNP IgE (Sigma) antibodies, respectively.  Cell morphology was also assessed by cytospin and staining with giemsa and toluidine blue.  To stimulate, cells were sensitised with anti-DNP IgE mAb (Sigma, 100 ng/ml) for 18 hours, washed twice to remove unbound IgE and stimulated with 20 ng/ml of DNP-HSA (Sigma) for 2 hours.


title: 2h post-infection of THP-1 NOT treated with SCOTS, replicate 6

source_name: cDNA obtained 2h post-infection

extract_protocol: TRIzol reagent (Gibco BRL) was used according to the manufacturer instruction. RNA was treated with DNase (Ambion). RNA sample was converted to cDNA in 5 independent reverse-transcription reactions as described previously (Graham, J. 1999. PNAS 96:11554-559).


title: Macrophages_unstimulated_rep2

description: Mature peripheral blood CD14+ monocyte-derived macrophages differentiated using GM-CSF over 11 days, unstimulated.

extract_protocol:
Trizol was used for total RNA extraction.
cRNA was prepared according to methods for small RNA samples (Baugh et al,
Nucleic acids research 29:E29, 2001). 500-1000 ng of RNA was reverse transcribed to cDNA using a poly(T) primer containing a T7-(dT)24 (Geneworks, Australia).

treatment_protocol: Macrophages were differentiated from isolated monocytes in the presence of GM-CSF. Briefly, peripheral blood was obtained from healthy volunteers and diluted 1:2 with PBS (GibcoBRL, New York, USA). Mononuclear leukocytes were isolated using a Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient and washed twice with PBS containing 2% FCS and 2mM EDTA. Monocytes were selected using anti-CD14 magnetic beads, according to the manufacturer’s protocol (Miltenyi BioTec, Bergisch Gladbach, Germany). The purified monocytes were resuspended at a cell concentration of 1x106 cells/ml in complete medium supplemented with 500 U/ml GM-CSF (BD PharMingen, Franklin Lakes, USA) and cultured for 11 days. Cultures were fed every 3 days by removing part of the supernatant and adding back fresh supplemented media. Adherent cells showed the typical morphology of macrophages.

tag: Macrophages differentiated from isolated CD14+ monocytes in the presence of GM-CSF, unstimulated.


title: Immature dendritic cells_rep2

description: Immature peripheral blood monocyte-derived dendritic cells. Differentiated using IL-4 and GM-CSF.

treatment_protocol: All cells were cultured in RPMI, 10% heat inactivated fetal calf serum, penicillin/streptomycin. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood via Ficoll gradient centrifugation. Dendritic cells were generated from human PBMCs as previously described (Sallusto et al., J. Exp Med 179:1109-1118, 1994). In brief, CD14+ monocytes were isolated using magnetic sorting (MACS, Miltenyi), and cultured for 5 days at 1X10^6 cells/ml in media containing IL-4 (800 U/ml) and GM-CSF (1500 U/ml). Immature DCs were harvested and resuspended in fresh media containing IL-4 and GM-CSF at 1X10^6 cells/ml/well in 24 well plates

tag: Human peripheral blood dendritic cells differentiated from CD14+ peripheral blood monocytes using IL-4 and GM-CSF, immature


title: 2h post-infection of THP-1 NOT treated with SCOTS, replicate 4

tag: Salmonella typhi infection of THP-1 cells, recovered 2 h post-infection


title: 2h post-infection of THP-1 NOT treated with SCOTS, replicate 3

tag: Genomic DNA from Salmonella typhi ISP1820


title: Macrophages_unstimulated_rep1

description: Mature peripheral blood CD14+ monocyte-derived macrophages differentiated using GM-CSF over 11 days.

tag: Macrophages differentiated from isolated CD14+ monocytes in the presence of GM-CSF, unstimulated


title: 2h post-infection of THP-1 NOT treated with SCOTS, replicate 5


title: Female Testosterone-Treated Submandibular Gland Sample B Technical Replicate

description: Young adult female BALB/c mice (n = 30), ovariectomized at age 8 weeks, were purchased from Taconic Laboratories (Germantown, NY) and maintained in constant temperature rooms with fixed light/dark periods of 12 hours. Animals were allowed to recover from surgery for 10 days, then anesthetized with ketamine and xylazine, and subcutaneous implants of placebo (n = 15; cholesterol, methylcellulose, lactose)- or testosterone (n = 15; 10 mg)-containing pellets were inserted in the subscapular region. Hormone pellets were obtained from Innovative Research of America (Sarasota, FL) and were designed to maintain male physiologic amounts of testosterone over a two week period. Following two weeks of hormone exposure, the mice were sacrificed in a CO2 chamber, and the lacrimal, meibomian, and submandibular glands were removed and pooled (5 mice per group) into three groups by treatment (n = 6 groups). Glands were immediately frozen and stored in liquid nitrogen until RNA isolation.

data_processing: Affymetrix data were filtered for spot quality by restricting analysis to genes with signal intensity greater than 100 for every sample.

label: Affymetrix labelling kit.

tag: Series includes pooled (n = 5 mice per biological replicate) samples from lacrimal, meibomian, and submandibular glands of female palcebo- and testosterone-treated BALB/c mice. All mice were ovariectomized at 8 weeks and allowed to recover from surgery for 10 days prior to hormone implanatation. All experiments were run in triplicate (pooled biological replicates) on Affymetrix MG U74A v.2 microarrays.


title: Female Testosterone-Treated Submandibular Gland Sample C Technical Replicate


title: Cord blood-derived mast cells_IgE 2h_rep2

description: ~95% pure population of human cord blood-derived mast cells differentiated using IL-10, IL-6 and stem cell factor, IgE-stimulated for 2 hours

treatment_protocol:
Mast cells were derived from cord blood using an established method (Ochi et al., J. Exp Med. 190: 267-280, 1999). Briefly, mononuclear leukocytes were isolated using a Ficoll Paque (Pharmacia Biotech AB, Uppsala, Sweden) density gradient. Cells were then seeded at 2 x 10^6 cells/ml of complete media supplemented with 100 ng/ml stem cell factor (SCF) (a gift from Amgen), 10 ng/ml interleukin (IL)-10 (BD), and 5 ng/ml IL-6 (BD). Mast cell cultures were passaged and transferred to new flasks weekly at a concentration of 10^6 cells/ml of media containing SCF/IL-10/IL-6 (described above). Mast cell maturity was assessed by metachromatic staining with 1%Toluidine blue (Sigma) in 0.5M HCl which specifically stains mast cell granules. Mast cells were used after 7-10 weeks of culture when cells were up to 95% pure and mature as determined by Toluidine Blue staining.
CBMCs were first primed with 2 μg/ml human IgE anti-DNP (Serotec) in the presence of 100 ng/ml SCF for 18 hours and then activated with 5 μg/ml mouse anti-human IgE (Serotec) with 100 ng/ml SCF for 2 hours by crosslinking FcεRI receptors. Control cells were incubated with complete media containing SCF. 3-6 x 106 activated and control mast cells were harvested by centrifugation and washed with PBS before RNA extraction.

tag: Human cord blood-derived mast cells differentiated over 7-10 weeks using IL-10, IL-6 and stem cell factor, IgE-stimulated for 2 hours


title: Neutrophils_unstimulated_rep1

treatment_protocol:
Fresh blood (50 ml) was collected in EDTA and used immediately.  Whole blood was centrifuged at 300 xg for 20 minutes, at room temperature.  The upper plasma layer was removed from the cell pellet and centrifuged at 1800 xg for 15 minutes, at room temperature, to remove platelets.  An equal volume of 1x PBS was used to replace the volume of plasma removed before red blood cells were sedimented in 0.6% dextran T500.  After 30 to 45 minutes, the upper leukocyte-rich phase was carefully transferred to a new tube and centrifuged at 170 xg for 5 minutes, to further remove platelets.  The cell pellet was then resuspended in platelet-poor plasma before carefully overlaying onto a 70% and 80% discontinuous isotonic Percoll gradient.

The cells were centrifuged through the discontinuous Percoll gradient at 900 xg for 30 minutes, at room temperature.  To avoid disturbing the gradient, the brakes were turned off.  After centrifugation, the granulocyte population, consisting mostly of neutrophils and eosinophils, were removed from the 70/80% interface.  To minimise neutrophil apoptosis, the cells were washed three times in unchilled wash buffer and centrifuged at room temperature.  For the first wash, the cells were centrifuged at 170 xg, for 10 minutes, to remove Percoll.  The supernatant was discarded and the pellet resuspended again in 50 ml of wash buffer before centrifugation at 170 xg, for 5 minutes.  The cells were then resuspended in wash buffer and counted on a haemocytometer.  To determine the number of contaminating eosinophils, the cells were cytocentrifuged onto a slide and stained with Wright's modified giemsa stain, according to the manufacturer's protocol, and 300 cells were counted.

To avoid neutrophil apoptosis, MACS was performed at room temperature.  Supernatant containing anti-CCR3 monoclonal antibodies (mAb) (10 mg/ml) was collected from hybridoma cultures.  After washing, the granulocyte cell pellet was resuspended in anti-CCR3 mAb supernatant at a concentration of 4 μg per 106 CCR3 positive cells, determined empirically by counting the percentage of eosinophils in the granulocyte sample.  The granulocytes were incubated in hybridoma supernatant for 10 minutes, at room temperature.  To wash off excess mAb, the cells were washed twice in wash buffer by centrifuging at 300 xg for 5 minutes.  The cells were then resuspended in 80 μL of wash buffer, and 20 μL of goat anti-mouse IgG microbeads (Miltenyi Biotec), per 10^7 total cells and incubated for 5 minutes at room temperature.  After incubation, the cells were washed twice in wash buffer before they were put through a negative selection column (Miltenyi Biotec), according to the manufacturer's protocols.
The flow-through was collected and the number of neutrophils and cell viability was determined by counting trypan blue stained cells on a hemocytometer.  Cell viability according to trypan blue was >98%.  To determine the sample purity, the cells were cytocentrifuged onto slides and stained with Wright's modified giemsa stain, and 300 cells were counted.  Unactivated neutrophils (high CD62L) were also detected by FACS analysis using a phycoerythrin-conjugated anti-human CD62L antibody (Pharmingen).  Only samples with >97 % neutrophils (high CD62L) were used for microarray analysis.  The main contaminating cells were eosinophils.  No monocytes were detected on cytospin slides.


title: Immature dendritic cells_rep1


title: 2h post-infection of THP-1 treated with 1 rounds of SCOTS, replicate 4

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, cells were washed with PBS and fresh RPMI containing 100 ug ml-1 was added to the wells. After 2 hours, cells were washed with PBS and recovered in 5 ml PBS. After centrifuation, the pellet was lysed with 2 ml TRIzol (Invitrogen).


title: Female Testosterone-Treated Submandibular Gland Sample A Technical Replicate


title: 2h post-infection of THP-1 treated with 1 rounds of SCOTS, replicate 1


title: Cord blood-derived mast cells_IgE 2h_rep1

treatment_protocol: Mast cells were derived from cord blood using an established method (Ochi et al., J. Exp Med. 190: 267-280, 1999). Briefly, mononuclear leukocytes were isolated using a Ficoll Paque (Pharmacia Biotech AB, Uppsala, Sweden) density gradient. Cells were then seeded at 2 x 10^6 cells/ml of complete media supplemented with 100 ng/ml stem cell factor (SCF) (a gift from Amgen), 10 ng/ml interleukin (IL)-10 (BD), and 5 ng/ml IL-6 (BD). Mast cell cultures were passaged and transferred to new flasks weekly at a concentration of 10^6 cells/ml of media containing SCF/IL-10/IL-6 (described above). Mast cell maturity was assessed by metachromatic staining with 1%Toluidine blue (Sigma) in 0.5M HCl which specifically stains mast cell granules. Mast cells were used after 7-10 weeks of culture when cells were up to 95% pure and mature as determined by Toluidine Blue staining.


title: Dendritic cells_LPS 48h_rep1

description: Mature peripheral blood monocyte-derived dendritic cells. Differentiated using IL-4 and GM-CSF and stimulated with LPS for 48 hours

treatment_protocol: All cells were cultured in RPMI, 10% heat inactivated fetal calf serum, penicillin/streptomycin.  Peripheral blood mononuclear cells (PBMCs) were isolated from human blood via Ficoll gradient centrifugation. Dendritic cells were generated from human PBMCs as previously described (Sallusto et al., J. Exp Med 179:1109-1118, 1994). In brief, CD14+ monocytes were isolated using magnetic sorting (MACS, Miltenyi), and cultured for 5 days at 1X10^6 cells/ml in media containing IL-4 (800 U/ml) and GM-CSF (1500 U/ml).  Immature DCs were harvested and resuspended in fresh media containing IL-4 and GM-CSF at 1X10^6 cells/ml/well in 24 well plates, then stimulated for 48hrs with LPS (100ng/ml) to induce maturation.

tag: Human peripheral blood dendritic cells differentiated from CD14+ peripheral blood monocytes using IL-4 and GM-CSF, then stimulated for 48 hours with LPS to induced maturation


title: Female Placebo-Treated Submandibular Gland Sample A Technical Replicate


title: Female Placebo-Treated Submandibular Gland Sample C Technical Replicate


title: Female Placebo-Treated Submandibular Gland Sample B Technical Replicate


title: 2h post-infection of THP-1 NOT treated with SCOTS, replicate 2


title: 2hr top replicate 3

status: Public on Jan 09 2006

city: Columbia

first: Emmanuel

last: Liscum

state: MO

address: Rollin Rd

department: Biological Sciences

institute: University of Missouri

description: wild type, gravity treated Brassica hypocotyl tissue

data_processing: GCOS 3.0

organism: Brassica oleracea

source_name: hypocotyl

label_protocol: Approximately 20 µg of total RNA was processed to produce biotinylated cRNA targets.

extract_protocol: Approximately 100 3-day-old B. oleracea seedlings that has been gravity stimulated were trisected into top flanks, vasculature, and bottom  flanks. The top and bottom flanks were collected and Total RNA was extracted from the tissue  using an RNeasy kit (Qiagen).

tag: wild type, 3 day old seedling


title: Eosinophils_control_rep1

description: 97% pure population of human peripheral blood eosinophils purified by Percoll and magnetic cell sorting (MACS) using anti-CD16, unstimulated

treatment_protocol:
Fresh blood (200 ml) was collected with EDTA and used immediately.  Red blood cells were sedimented from whole blood in 0.6% dextran T500 (Pharmacia) for 30 minutes, at room temperature.  Once red blood cells had sedimented to the bottom, the upper leukocyte-rich phase was carefully transferred to a new tube by pipetting.  The leukocyte-rich phase was diluted 1:2 with wash buffer before overlaying onto a 70% and 80% discontinuous isotonic Percoll gradient.

Cells were centrifuged through the Percoll gradient at 1300 xg for 30 minutes, at room temperature.  To avoid disturbing the gradient, brakes were turned off.  After centrifugation, the granulocyte population, consisting mostly of neutrophils and eosinophils, were removed from the 70/80% interface.  To minimise eosinophil activation, the cells were washed twice in cold wash buffer and centrifuged at 4°C.  For the first wash, the cells were centrifuged at 680 xg, for 7 minutes, to remove Percoll.  The supernatant was discarded and the pellet resuspended again in 50 ml of wash buffer before centrifugation at 470 xg, for 5 minutes.  To remove the low numbers of contaminating red blood cells, the cell pellet was resuspended in 2 ml of 1x red blood cell lysis buffer and centrifuged immediately.  After washing once in 2 ml of 1x lysis kit wash buffer, the cell pellet was resuspended in wash buffer and the cells were counted on a haemocytometer.

Neutrophils were depleted from the granulocyte population using anti-CD16 magnetic beads, according to the manufacturer's protocol (Miltenyi BioTec, Bergisch Gladbach, Germany).  After the granulocytes were counted, they were resuspended in MACS buffer (50 μl/5 × 107 total cells).  Anti-CD16 magnetics beads (Miltenyi Biotec) were also added (50 μl/5 × 107 total cells).  Cells were incubated with anti-CD16 magnetic beads at 6–12°C for 30–40 minutes. Depletion columns and flow resistors were used, according to the manufacturer's protocol (Miltenyi Biotec).  The columns were assembled onto a Varios MACS magnetic apparatus (Miltenyi Biotec) and washed with MACS buffer. After incubation, the cell suspension was diluted with MACS buffer (5 × 107 total cells/ml) before applying to the column.  CD16 positive cells, including neutrophils, were retained in the column by the magnetic force.  Eosinophils were collected into a chilled 15 ml tube on ice.  Eosinophils were stained with trypan blue and counted on a haemocytometer.  Cell viability according to trypan blue exclusion was >97%. To determine the purity of the sample, the cells were cytocentrifuged onto a slide and stained with Wright's modified Giemsa stain (Sigma Diagnostics), according to the manufacturer's instructions.  At least 400 cells were counted on each slide.  Only samples with >97% eosinophils were used in further experiments.  The main contaminating cells were neutrophils.

tag: Human peripheral blood purified eosinophils (unstimulated)


title: unstimulated control replicate 3

description: wild type, unstimulated hypocotyl Brassica hypocotyl tissue

extract_protocol: Approximately 100 3-day-old dark grown, unstimulated B. oleracea seedlings were trisected into left and right flanks and vasculature. The flanks were collected and Total RNA was extracted from the tissue  using an RNeasy kit (Qiagen).


title: Cord blood-derived mast cells_control_rep1

description: ~95% pure population of human cord blood-derived mast cells differentiated using IL-10, IL-6 and stem cell factor, unstimulated

tag: Human cord blood-derived mast cells differentiated over 7-10 weeks using IL-10, IL-6 and stem cell factor, unstimulated


title: unstimulated control replicate 2


title: 2hr top replicate 2


title: Eosinophils_PMA 2h_rep1

description: 97% pure population of human peripheral blood eosinophils purified by Percoll and magnetic cell sorting (MACS) using anti-CD16, PMA-stimulated for 2 hours

treatment_protocol:
Fresh blood (200 ml) was collected with EDTA and used immediately. Red blood cells were sedimented from whole blood in 0.6% dextran T500 (Pharmacia) for 30 minutes, at room temperature. Once red blood cells had sedimented to the bottom, the upper leukocyte-rich phase was carefully transferred to a new tube by pipetting. The leukocyte-rich phase was diluted 1:2 with wash buffer before overlaying onto a 70% and 80% discontinuous isotonic Percoll gradient.

Cells were centrifuged through the Percoll gradient at 1300 xg for 30 minutes, at room temperature. To avoid disturbing the gradient, brakes were turned off. After centrifugation, the granulocyte population, consisting mostly of neutrophils and eosinophils, were removed from the 70/80% interface. To minimise eosinophil activation, the cells were washed twice in cold wash buffer and centrifuged at 4°C. For the first wash, the cells were centrifuged at 680 xg, for 7 minutes, to remove Percoll. The supernatant was discarded and the pellet resuspended again in 50 ml of wash buffer before centrifugation at 470 xg, for 5 minutes. To remove the low numbers of contaminating red blood cells, the cell pellet was resuspended in 2 ml of 1x red blood cell lysis buffer and centrifuged immediately. After washing once in 2 ml of 1x lysis kit wash buffer, the cell pellet was resuspended in wash buffer and the cells were counted on a haemocytometer.

Neutrophils were depleted from the granulocyte population using anti-CD16 magnetic beads, according to the manufacturer's protocol (Miltenyi BioTec, Bergisch Gladbach, Germany). After the granulocytes were counted, they were resuspended in MACS buffer (50 μl/5 × 107 total cells). Anti-CD16 magnetics beads (Miltenyi Biotec) were also added (50 μl/5 × 107 total cells). Cells were incubated with anti-CD16 magnetic beads at 6–12°C for 30–40 minutes. Depletion columns and flow resistors were used, according to the manufacturer's protocol (Miltenyi Biotec). The columns were assembled onto a Varios MACS magnetic apparatus (Miltenyi Biotec) and washed with MACS buffer. After incubation, the cell suspension was diluted with MACS buffer (5 × 107 total cells/ml) before applying to the column. CD16 positive cells, including neutrophils, were retained in the column by the magnetic force. Eosinophils were collected into a chilled 15 ml tube on ice. Eosinophils were stained with trypan blue and counted on a haemocytometer. Cell viability according to trypan blue exclusion was >97%. To determine the purity of the sample, the cells were cytocentrifuged onto a slide and stained with Wright's modified Giemsa stain (Sigma Diagnostics), according to the manufacturer's instructions. At least 400 cells were counted on each slide. Only samples with >97% eosinophils were used in further experiments. The main contaminating cells were neutrophils.
The purified eosinophils were resuspended at a cell concentration of 2.5 × 106 cells/ml in RPMI1640 medium (GibcoBRL), supplemented with 10% heat-inactivated FCS, 0.5 U/ml penicillin, 0.5 μg/ml streptomycin and 2mM L-glutamine.  Cells were incubated for 2 hours in 24-well culture plates (Nalge Nunc International, Denmark) at 37°C in 5% CO2, with phorbol-12-myristate-13-acetate (PMA) (50 ng/ml).

tag: Human peripheral blood purified eosinophils (PMA-stimulated for 2 hours)


title: 2h post-infection of THP-1 NOT treated with SCOTS, replicate 1

description: This sample was used to analyse the effect of SCOTS on transcript population.


title: 2hr top replicate 1


title: unstimulated control replicate 1


title: 2hr bottom replicate1

description: wild type, gravitytreated Brassica hypocotyl tissue

extract_protocol: Approximately 100 3-day-old B. oleracea seedlings that has been gravity stimulated were trisected into top flanks, vasculature, and bottom flanks. The top and shaded flanks were collected and Total RNA was extracted from the tissue  using an RNeasy kit (Qiagen).


title: 2hr bottom replicate 2

extract_protocol: Approximately 100 3-day-old B. oleracea seedlings that has been gravity stimulated were trisected into top flanks, vasculature, and bottom flanks. The  top and shaded flanks were collected and Total RNA was extracted from the tissue  using an RNeasy kit (Qiagen).


title: Supernatant of infection treated with 3 rounds of SCOTS, replicate 6

description: This sample was used to analyse the expression profiles of Salmonella typhi during infection of human macrophages (THP-1)

source_name: 3XSCOTS cDNA obtained from infection supernatant

treatment_protocol: 2E7 THP-1 cells (treated with 1E-7 M phorbol 12-myristate 13-acetate) were infected with Salmonella typhi at a multiplicity of infection of 10:1. At time 0, the supernatant was removed and centrifuge to recover the bacteria. The pellet was lysed with 2 ml of TRIzol (Gibco BRL)

tag: Salmonella typhi infection of THP-1 cells, recovered from the culture medium supernatant


title: 2hr lit replicate 3

description: wild type, blue-light treated Brassica hypocotyl tissue

extract_protocol: Approximately 100 3-day-old B. oleracea seedlings that has been stimulated with unidirectional blue-light were trisected into lit flanks, vasculature, and shaded flanks. The shaded flanks were collected and Total RNA was extracted from the tissue  using an RNeasy kit (Qiagen).