title: Germ Cell Tumor 401A

source_name: Genomic DNA from germ cell tumor 401A labeled with Cy3


title: Germ Cell Tumor 396A

status: Public on Nov 15 2007

type: genomic

city: New York

first: James

last: Korkola

state: NY

address: 1275 York Ave

department: Cell Biology

country: USA

institute: Memorial Sloan-Kettering

description: abbreviations: EC- embryonal carcinoma; CC- choriocarcinoma; Sem- Seminoma; YST- yolk sac tumor; Ter- teratoma; Ter(I)- immature teratoma; Ter(M)- mature teratoma; TMT- teratoma with malignant transfomation; trophs- trophoblast cells; rplnd- retroperitoneal lymph node; LN- lymph node

hyb_protocol: Labeled test and reference DNA were mixed and hybridized overnight in Spectral Hybridization buffer (Spectral Genomics) at 37C under lifter slips (Erie Scientific); following morning slides were washed sequentially according to array manufacturer's recommendations (Spectral Genomics)

scan_protocol: Slides were scanned on an Axon 4000B scanner Images were quantified using Genepix Pro v.3

data_processing: Utilized difference between log2 median Cy3 and Cy5 values as raw data for analysis; Subarray (block) median normalization and global loess correction was performed and duplicates were averaged; Changes in copy number were identified by CBS algorithm (see Chen W. et al., Blood 107(6):2477-85, 2006)

label: Cy5 & Cy3

molecule: genomic DNA

organism: Homo sapiens

source_name: Genomic DNA from germ cell tumor 396A labeled with Cy3 and Cy5 (dye swap)

label_protocol: DPNII digested DNA was labeled using a Bioprime Labeling Kit (Invitrogen) incorporating fluorescent dye (either dCTP-Cy3 or dCTP-Cy5) and mixed with differentially labeled reference DNA

extract_protocol: DNA was extracted using previously described protocols (see Chen W. et al., Blood 107(6):2477-85, 2006)

tag: germ cell tumor

tag: Histology

value: EC,Ter(M)

tag: Site

value: Axillary LN

label: Cy3 & Cy5

source_name: placenta reference DNA

tag: karyotypically normal female placental DNA


title: Germ Cell Tumor 385A

source_name: Genomic DNA from germ cell tumor 385A labeled with Cy3

value: Ter(M)

value: rplnd


title: Germ Cell Tumor 373A

source_name: Genomic DNA from germ cell tumor 373A labeled with Cy3

value: EC,YST

value: Testes


title: Germ Cell Tumor 399A

source_name: Genomic DNA from germ cell tumor 399A labeled with Cy3

value: EC

value: testis


title: Germ Cell Tumor 372B

source_name: Genomic DNA from germ cell tumor 372B labeled with Cy3

value: YST,trophs

value: Mediastinum


title: Germ Cell Tumor 371A

source_name: Genomic DNA from germ cell tumor 371A labeled with Cy3


title: Germ Cell Tumor 362A

source_name: Genomic DNA from germ cell tumor 362A labeled with Cy5

value: Sem


title: Germ Cell Tumor 354B

source_name: Genomic DNA from germ cell tumor 354B labeled with Cy3

value: Ter(M),sarcoma


title: Germ Cell Tumor 287B

source_name: Genomic DNA from germ cell tumor 287B labeled with Cy3

value: Sem,Ter(M,I),YST


title: Germ Cell Tumor 318D

source_name: Genomic DNA from germ cell tumor 318D labeled with Cy3


title: Germ Cell Tumor 310A

source_name: Genomic DNA from germ cell tumor 310A labeled with Cy3

value: Sem,YST,EC


title: Germ Cell Tumor 288A

source_name: Genomic DNA from germ cell tumor 288A labeled with Cy3

value: YST


title: Germ Cell Tumor 289A

source_name: Genomic DNA from germ cell tumor 289A labeled with Cy3

value: Lung


title: Germ Cell Tumor 303A

source_name: Genomic DNA from germ cell tumor 303A labeled with Cy5

value: adrenal gland


title: Germ Cell Tumor 299A

source_name: Genomic DNA from germ cell tumor 299A labeled with Cy3

value: Ter(M),YST,EC


title: Germ Cell Tumor 268A

source_name: Genomic DNA from germ cell tumor 268A labeled with Cy3

value: EC,Ter(M)


title: Germ Cell Tumor 256A

source_name: Genomic DNA from germ cell tumor 256A labeled with Cy3


title: Germ Cell Tumor 248A

source_name: Genomic DNA from germ cell tumor 248A labeled with Cy3

value: Ter

source_name: placenta reference DNA


title: Germ Cell Tumor 249A

source_name: Genomic DNA from germ cell tumor 249A labeled with Cy3

value: CC


title: KO_sham_mid_2

status: Public on Nov 16 2007

type: RNA

city: Perth

first: Sulev

last: Koks

state: Western Australia

address: 90 South Street

department: Centre for Molecular Medicine and Innovative Therapeutics

country: Australia

institute: Murdoch University

hyb_protocol: Following fragmentation, 4 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse 430A 2.0 Gene Expression Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Affymetrix Scanner 3000

data_processing: The raw intensity data (cel files) were analyzed with dChip2005 using invariant-set normalization and the PM-MM model

label: biotin

molecule: total RNA

organism: Mus musculus

source_name: midbrain

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

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.


title: Germ Cell Tumor 243A

source_name: Genomic DNA from germ cell tumor 243A labeled with Cy3

value: YST,TMT

value: rplnd


title: Germ Cell Tumor 208B

source_name: Genomic DNA from germ cell tumor 208B labeled with Cy3

value: EC,YST,Ter


title: Germ Cell Tumor 231A

source_name: Genomic DNA from germ cell tumor 231A labeled with Cy3


title: Germ Cell Tumor 228A

source_name: Genomic DNA from germ cell tumor 228A labeled with Cy3


title: Germ Cell Tumor 224A

source_name: Genomic DNA from germ cell tumor 224A labeled with Cy3

value: EC,YST,CC

value: Testes


title: Germ Cell Tumor 221A

source_name: Genomic DNA from germ cell tumor 221A labeled with Cy3


title: Germ Cell Tumor 220A

source_name: Genomic DNA from germ cell tumor 220A labeled with Cy3


title: Germ Cell Tumor 214A

source_name: Genomic DNA from germ cell tumor 214A labeled with Cy3

value: EC,YST


title: Germ Cell Tumor 210A

source_name: Genomic DNA from germ cell tumor 210A labeled with Cy3


title: Germ Cell Tumor 202A

source_name: Genomic DNA from germ cell tumor 202A labeled with Cy3

value: EC,YST,Ter(M),Trophs


title: Germ Cell Tumor 215A

source_name: Genomic DNA from germ cell tumor 215A labeled with Cy3


title: Germ Cell Tumor 192A

source_name: Genomic DNA from germ cell tumor 192A labeled with Cy3

value: Sem

value: testis


title: Germ Cell Tumor 198A

source_name: Genomic DNA from germ cell tumor 198A labeled with Cy5

value: CC,EC


title: Germ Cell Tumor 193A

source_name: Genomic DNA from germ cell tumor 193A labeled with Cy3

value: YST,Ter(I),Sem,Trophs


title: KO_sham_med_1

source_name: medulla


title: Germ Cell Tumor 187A

source_name: Genomic DNA from germ cell tumor 187A labeled with Cy3

source_name: placenta reference DNA


title: Germ Cell Tumor 186B

source_name: Genomic DNA from germ cell tumor 186B labeled with Cy3 and Cy5 (dye swap)

value: YST

value: Mediastinum


title: Germ Cell Tumor 186A

source_name: Genomic DNA from germ cell tumor 186A labeled with Cy3

value: mediastinum


title: Germ Cell Tumor 208A

source_name: Genomic DNA from germ cell tumor 208A labeled with Cy3


title: Germ Cell Tumor 177B

source_name: Genomic DNA from germ cell tumor 177B labeled with Cy3


title: Germ Cell Tumor 155C

source_name: Genomic DNA from germ cell tumor 155C labeled with Cy3

value: EC,Ter

value: liver


title: Germ Cell Tumor 155A

source_name: Genomic DNA from germ cell tumor 155A labeled with Cy3

value: EC, Ter


title: Germ Cell Tumor 052B

source_name: Genomic DNA from germ cell tumor 052B labeled with Cy3

value: EC, Sem


title: Germ Cell Tumor 171A

source_name: Genomic DNA from germ cell tumor 171A labeled with Cy3

value: EC, Ter(I,M), TMT


title: nomad 06

city: Raleigh

first: Youssef

last: Idaghdour

state: NC

address: South Gardner Hall

department: Genetics

institute: NCSU

hyb_protocol: Illumina hybridization recommencded protocol was followed.

scan_protocol: The BeadChips were imaged using Illumina’s BeadArray Reader

data_processing: The raw intensities were extracted using the Gene Expression Module in Illumina’s BeadStudio software

source_name: Total leukocytes

label_protocol: cRNA labelling using Cy3 was performed using the Illumina TotalPrep RNA Amplification kit (Ambion, Austin, TX) following the manufacturer’s instructions.

extract_protocol: Total leukocytes were isolated from 9 ml whole blood and its total RNA stabilized and extracted using the LeukoLOCK™ Total RNA Isolation System (Ambion, Austin, TX)

tag: SEX

value: Female

tag: Lifestyle

value: Nomad


title: nomad 12

value: Male


title: rural 04

value: Rural


title: urban 03

value: Urban


title: urban 16


title: urban 01


title: urban 11


title: rural 01


title: urban 10


title: rural 11


title: nomad 02


title: nomad 03


title: urban 15


title: urban 09


title: nomad 01


title: rural 08


title: nomad 08


title: nomad 10


title: nomad 11


title: urban 08


title: nomad 09


title: nomad 07


title: nomad 05


title: urban 05


title: urban 04


title: rural 02


title: urban 14


title: rural 10


title: rural 03


title: urban 13


title: rural 09


title: urban 02


title: urban 12


title: nomad 04


title: urban 18


title: nomad 15


title: rural 07


title: rural 12


title: rural 06


title: nomad 14


title: urban 07


title: AGD lesion fish 6 (IF10L) vs Control fish 6 (CF10)

city: Victoria

first: Ben

middle: F

last: Koop

state: BC

address: PO Box 3020 STN CSC

department: Biology

country: Canada

institute: The University of Victoria

hyb_protocol: Post-print processing of arrays was done by washing 2X  5 min in 0.2% SDS, 5X 1 min in MilliQ water followed by immersion in MilliQ water at 100°C for 3 min. The slides were dried by centrifugation at 512 X g for 5 min.Pre-hybridization involved placing the arrays in (5X SSC, 1% SDS and 3% BSA (Sigma)) and incubating at 49°C in a water bath for 90 min. Thereafter, the arrays were washed thrice for 20 sec in MilliQ water and dried by centrifugation as above. For hybridization, 500 ng of labeled target reconstituted to a final volume of 26 µL, 30 µL of 2X formamide hybridization buffer (Genisphere) and 4 µL LNT dT blocker (Genisphere) were used. Hybridization was done at 49°C in a water bath for 16hrs. The arrays were washed first in (2X SSC, 0.1% SDS) for 10 min at 49°C, 2 X 5 min in (2X SSC, 0.1%SDS) at room temperature, 2 X 5 min in 1X SSC, 4 X 5 min in 0.1X SSC followed immediately by centrifugation at 512 X g for 5 min.

scan_protocol: Imaging was done at 10 µm resolution using ScanArray™ Express microarray scanner (Packard Bioscience). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively, at the same laser power (90%), with adjusted photomultiplier tube settings between slides to balance the Cy5 and Cy3 channels. Image analysis and fluorescent intensity data was extracted from Tiff images using ImaGene™ 5.6 Standard Edition software. Scanning was done using ScanArray™ Express microarray scanner.

data_processing: LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Genespring GX software was used.

organism: Salmo salar

source_name: Total RNA from AGD-affected gill lesion tissue at 36 days post inoculation with Neoparamoeba perurans

label_protocol:
2μg of total RNA from each fish was subjected to 1 round of amplification by using the Amino Allyl MessageAmp I aRNA amplification kit (Ambion)
Coupling of aRNA was performed using either Cy3 or Cy5 dye according to the Amino Ally MessageAMP™ I aRNA Amplification Kit (Ambion) protocol with minor modifications. Briefly, one vial of Cy3 or Cy5 reactive dyes (Amersham) was resuspended in 22µl of DMSO prior to coupling. 3µg of aRNA was dried to completion in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY).  The aRNA was resuspended in 4.5µl of coupling buffer. Thereafter, 5.5µl of dye was added to the aRNA:coupling buffer and incubated for 30 minutes in the dark. 2.25µl of 4M hydroxylalamine was then added to quench the reaction followed by 12.75µl of water to obtain a total volume to 30µl. Dye-labeled aRNA purification was performed according to the manufacturer’s instructions.

growth_protocol: Forty Atlantic salmon (Salmo salar) were reared in two 500L tanks (20 fish per tank). Fish were acclimated from freshwater to seawater (35 ppt) over a three week period then maintained in recirculated seawater at an average temperature of 15.5°C . Fish were fed with commercial pellets (Skretting, Australia).

extract_protocol: Total RNA extracted using RNEasy® kit (Qiagen) with on-column DNAse treatment

treatment_protocol: Amoebae, later confirmed to be Neoparamoeba perurans were harvested from the gills of moribound AGD-affected Atlantic salmon (Salmo salar). Twenty fish in one 500L tank were exposed to N. perurans at 500 cells L-1 while the other tank (20 fish)  became the control AGD-naive group. Fish were held for 36 days then fish were euthanised and the gill filaments of fish in both treatments were excised and stored in RNAlater (Sigma-Aldrich).

tag: AGD-affected fish 6, AGD lesion tissue, IF10L

tag: Dissected lesion tissue from AGD-affected gill arch

tag: Fish inoculated with Neoparamoeba perurans, the aetiological agent of AGD

tag: weight

source_name: Total RNA from AGD-naive gill non-lesion tissue

tag: AGD-naive fish 6, no lesion tissue, CF10

tag: Dissected non-lesion tissue from AGD-naive gill arch

tag: Weight


title: AGD lesion fish 1 (IF1L) vs AGD no lesion fish 1 (IF1NL)

tag: AGD-affected fish 1, AGD lesion tissue, IF1L

source_name: Total RNA from AGD-affected gill non-lesion tissue at 36 days post inoculation with Neoparamoeba perurans

tag: AGD-affected fish 1, AGD no lesion tissue, IF1NL

tag: Dissected non-lesion tissue from AGD-affected gill arch


title: nomad 16


title: urban 17


title: nomad 13


title: rural 05


title: Control fish 6 (CF10) vs AGD lesion fish 6 (IF10L)


title: Control fish 5 (CF9) vs AGD lesion fish 4 (IF6L)

tag: AGD-naive fish 5, no lesion tissue, CF9

tag: AGD-affected fish 4, AGD lesion tissue, IF6L


title: AGD lesion fish 5 (IF8L) vs AGD no lesion fish 5 (IF8NL)

tag: AGD-affected fish 5, AGD lesion tissue, IF8L

tag: AGD-affected fish 5, AGD no lesion tissue, IF8NL


title: AGD no lesion fish 1 (IF1NL) vs AGD lesion fish 1 (IF1L)


title: AGD lesion fish 1 (IF1L) vs Control fish 1 (CF2)

tag: AGD-naive fish 1, no lesion tissue, CF2


title: AGD lesion fish 4 (IF6L) vs Control fish 5 (CF9)


title: urban 06

tag: SEX


title: Control fish 3 (CF7) vs AGD lesion fish 3 (IF4L)

tag: AGD-naive fish 3, no lesion tissue, CF7

tag: AGD-affected fish 3, AGD lesion tissue, IF4L


title: Control fish 1 (CF2) vs AGD lesion fish 1 (IF1L)

tag: Dissected lesion tissue from AGD-affected gill arch


title: AGD no lesion fish 2 (IF3NL) vs AGD lesion fish 2 (IF3L)

tag: AGD-affected fish 2, AGD no lesion tissue, IF3NL

tag: Fish inoculated with Neoparamoeba perurans, the aetiological agent of AGD

tag: AGD-affected fish 2, AGD lesion tissue, IF3L


title: ar10 pdc1, 5, 6  quadruple mutant glc limited chemostat with phenylalanine-2

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

hyb_protocol: Hybridization protocol Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner.

scan_protocol: Scan protocol Scanning with the Affymetrix GeneArray Scanner.

label: Biotinylated dUTP - streptavidine Phycoerythrin (SAPE)

molecule: polyA RNA

organism: Saccharomyces cerevisiae

source_name: chemostat culture sample

label_protocol: Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual. Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual . Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner.

growth_protocol:
Chemostat cultivation.
Aerobic chemostat cultivation was performed at 30°C in 1-liter (working volume) laboratory fermentors (Applikon, Schiedam, The Netherlands) at a stirrer speed of 800 rpm and pH 5.0 with a dilution rate of 0.10 h_1. The pH was kept constant by usingan ADI 1030 biocontroller (Applikon) and automatic addition of 2 M KOH. The fermentor was flushed with air at a flow rate of 0.5 liter min_1 by using a Brooks 5876 mass flow controller (Brooks Instruments, Veenendaal, The Netherlands). The dissolved oxygen concentration was continuously monitored with an Ingold model 34 100 3002 probe (Mettler-Toledo, Greifensee, Switzerland) and was more than 50% of air saturation.
Carbon-limited steady-state chemostat cultures of both wild-type and mutant strains were grown on the mineral medium described by Verduyn et al. (1) containing 7.5 g of glucose liter_1 as carbon source and 5.0 g of phenylalanine liter_1 as the sole nitrogen source.
When phenylalanine was the sole nitrogen source, the amino acid solution was sterilized separately by autoclaving it before addition to the medium, and the absence of (NH4)2SO4 was compensated for by addition of equimolar amounts of K2SO4. For chemostat cultivation of pyruvate decarboxylase-negative strains, 7.1 g of glucose liter_1 and 0.38 g of acetate liter_1 (5% acetate on a carbon basis) were used as carbon sources to overcome the C2 requirement of PDC negative
strains (2).
1- Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:395–403.
2- Flikweert, M. T., L. Van Der Zanden, W. M. Janssen, H. Y. Steensma, J. P. van Dijken, and J. T. Pronk. 1996. Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12:247–257.

extract_protocol:
Extracted molecule polyA RNA Extraction protocol Sampling and RNA Isolation—
Samples from the chemostat cultures were taken as rapidly as possible to limit any potential changes in transcript profiles during the procedure. 40–60 ml of culture broth was sampled directly from the chemostat into a beaker containing 200 ml of liquid nitrogen. With vigorous stirring, the sample froze instantly. The frozen sample was then broken into small fragments and transferred to a 50-ml centrifuge tube. The sample was then thawed at room temperature, ensuring that it remained as close to zero as possible. Cells were pelleted (5000 rpm at 0 °C for 4 min), resuspended in 2 ml of ice-cold AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0) and aliquoted into 5 Eppendorf tubes. This corresponded to _20 mg of dry weight per tube. For each array, total RNA was extracted from a single tube using the hot-phenol method  or the FastRNA kit, Red (BIO 101, Inc., Vista, CA).

treatment_protocol: Treatment protocol Liquid nitrogen quenching

tag: CENPK609-11A Mata  MAL2-8c SUC2 pdc1, 5, 6 ydr380w delta


title: AGD lesion fish 3 (IF4L) vs AGD no lesion fish 3 (IF4NL)

tag: weight

tag: AGD-affected fish 3, AGD no lesion tissue, IF4NL

tag: Weight


title: AGD no lesion fish 5 (IF8NL) vs AGD lesion fish 5 (IF8L)

tag: Dissected non-lesion tissue from AGD-affected gill arch


title: AGD lesion fish 5 (IF8L) vs Control fish 4 (CF8)

tag: AGD-naive fish 4, no lesion tissue, CF8

tag: Dissected non-lesion tissue from AGD-naive gill arch


title: aro10delta thi3delta strain glc-limited chemostat with phenylalanine -1

source_name: Chemostat culture sample

tag: CENPK632-3B Mat alpha MAL2-8c SUC2 ydl080c delta, ydr380w delta


title: AGD lesion fish 2 (IF3L) vs Control fish 2 (CF6)

tag: AGD-naive fish 2, no lesion tissue, CF6


title: ar10 thi3 double mutant glc limited chemostat with phenylalanine1

tag: MATa MAL2–8c SUC2 ydl080c delta ydr380w::loxP-Kan-loxP


title: aro10delta glc-limited chemostat with phenylalanine-2

tag: CEN.PK555-4A MATa MAL2–8c SUC2 ydr380w::loxP-Kan-loxP


title: aro10delta strain glc-limited chemostat with phenylalanine

source_name: chemostat sample

label_protocol: Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual . Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner.

tag: CEN.PK555-4A (MATa MAL2–8c SUC2 ydr380w::loxP-Kan-loxP)


title: aro10 pdc1, 5, 6 delta quadrple mutant glc-limited chemostat with phenylalanine-1

tag: Saccharomyces cerevisiae CENPK609-11A Mat a ydr380w, pdc1, 5, 6 delta,


title: AGD no lesion fish 3 (IF4NL) vs AGD lesion fish 3 (IF4L)

tag: AGD-affected fish 3, AGD lesion tissue, IF4L

tag: Dissected lesion tissue from AGD-affected gill arch


title: AGD lesion fish 3 (IF4L) vs Control fish 3 (CF7)

tag: Fish inoculated with Neoparamoeba perurans, the aetiological agent of AGD

tag: AGD-naive fish 3, no lesion tissue, CF7


title: Control fish 2 (CF6) vs AGD lesion fish 2 (IF3L)

tag: AGD-affected fish 2, AGD lesion tissue, IF3L


title: 2426 micro PSY rep_2

city: Zurich

first: Stefan

last: Zoller

address: Universitatstrasse 16

department: Genetic Diversity Centre

country: Switzerland

institute: ETH Zurich

description: B. japonicum regR mutant strain grown microaerobically

hyb_protocol: Modification of Affymetrix protocol used for P. aeruginosa

scan_protocol: Standard Affymetrix procedures

data_processing: GeneChip Operating Software (GCOS) version 1.1 (Affymetrix)

organism: Bradyrhizobium japonicum

label_protocol: 2.8 mg (free-living cultures) and 7.8 mg (nodules)  cDNA was biotin-labeled

growth_protocol: cells were grown to an OD600 0.4-0.5 in PSY medium; soybean nodules were harvested 13 and 21 days post inoculation

extract_protocol: RNA was extracted as described by Hauser et al. (2007) and Pessi et al.(2007)

treatment_protocol: no treatment was applied

tag: Genotype

tag: Growth condition

value: micro

tag: Medium

value: PSY


title: 2426 micro PSY rep_3

source_name: B. japonicum regR mutant strain grown microaerobically


title: 2426 micro PSY rep_5


title: 2426 micro PSY rep_1


title: 2426 micro PSY rep_4


title: Control fish 4 (CF8) vs AGD lesion fish 5 (IF8L)

tag: weight

tag: AGD-affected fish 5, AGD lesion tissue, IF8L

tag: Weight


title: 2426 soybean nodules 13 dpi  rep_3

description: Nodules soybean 13 dpi infected with B. japonicum regR mutant strain

value: nodules

value: soybean

tag: Time

value: 13 Days


title: 2426 soybean nodules 13 dpi  rep_2

source_name: Nodules soybean 13 dpi infected with B. japonicum regR mutant strain


title: Monocyte_HCMV_LY-3

city: Shreveport

first: Andrew

middle: D.

last: Yurochko

state: LA

address: 1501 Kings Highway

department: Microbiology & Immunology

institute: LSUHSC-S

description: HCMV-infected monocyte pretreated with LY, biological rep3

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human U95Av2 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling with a target intensity value of 2500 as normalization method.

source_name: HCMV-infected monocyte pretreated with LY at 4 hours post infection

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Monocytes were incubated in RPMI supplemented with 10% human AB serum at 37C and 5% CO2.

extract_protocol: RNA STAT-60 extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Isolated monocytes were pretreated nonadherently at 37C for 45 minutes with 5 μM of the NF-κB inhibitor Bay11-7082, 50 μM of the PI(3)K inhibitor LY294002, or dimethyl sulfoxide (DMSO) as the solvent control prior to infection.  Cells were then HCMV infected (MOI 15) and incubated nonadherently at 37C for 4 hours.

tag: Tissue

value: Peripheral blood

tag: Virus Strain

value: Towne/E


title: Whole genome recruitment of Rpb4 in S.cerevesiae

city: Bangalore

first: madavan

last: vasudevan

state: Karnataka

address: Kasturi Nagar

country: India

institute: Theomics International Private Limited

description: Microarray slides were obtained from Agilent Technologies (yeast 4 X 44k, whole genome array). The arrays comprise of 60 mer oligo nucleotides probes at ~ 290 bp spacing, covering ~12 Mb of the genome.

hyb_protocol: IPDNA and WCE was hybridized according to Agilent recommended protocol Reference: Yeast ChIP on chip protocol, Agilent Technologies (version 9.1).

scan_protocol: Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.

data_processing: Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol for array CGH that showed high correlation (r2=0.9) of bound probes between data of the two biological replicates. Identification of bound probes and genes was made using Agilent ChIP analytics software (version 1.3) by using Whitehead Institute Per-array Neighbourhood model v1.0. In the neighbourhood model, if candidate bound probe set p-value was less than 0.05, the central probe was marked as potentially bound, and the probe sets were required to pass one of two additional filters: the center probe in the probe set has a single probe p-value less than 0.05 or one of the flanking probes has a single point p-value less than 0.25. These two filters cover situations where a binding event occurs midway between two probes and each detects the event, weakly.

source_name: S.cerevisiae log phase culture cells - Immunoprecipitate

label_protocol: IPDNA was labeled according to Agilent recommended protocol Reference: Yeast ChIP on chip protocol, Agilent Technologies (version 9.1).

extract_protocol: TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25 C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. Two hundred microliters of whole-cell extract was incubated with Rabbit IgG agarose beads (Sigma) for 3 hrs at 4°C with agitation. Beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of lysis buffer plus 500 mM NaCl, twice with 1 ml of wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA) and once with 1 ml of Tris-EDTA (TE; 10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Bound material was eluted from the beads by resuspending beads in 100 µl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and incubating 10 min at 65°C with occasional agitation. Samples were centrifuged briefly and the cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 30 µl of TE. These samples were further purified with Qiagen PCR product purification Kit.

tag: Organism

tag: Strain

value: S288c, TAP tagged Rpb4

tag: Growth phase

value: Log

value: YPD

source_name: S.cerevisiae log phase culture cells - Whole cell extract

label_protocol: WCE was labeled according to Agilent recommended protocol Reference: Yeast ChIP on chip protocol, Agilent Technologies (version 9.1).

extract_protocol: TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25 C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. 50 ul of WCE was mixed with 150 µl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 50 µl of TE. These samples were further purified with Qiagen PCR product purification Kit.


title: Whole genome recruitment of Rpb4 in S.cerevesiae Replicate


title: 2426 soybean nodules 13 dpi  rep_5


title: 2426 soybean nodules 13 dpi  rep_1


title: 2426 soybean nodules 21 dpi  rep_5

description: Nodules soybean  21 dpi infected with B. japonicum regR mutant strain

value: 21 Days


title: Whole genome recruitment of Rpb3 in S.cerevesiae Replicate

value: S288c, TAP tagged Rpb3


title: Spo11-18myc ChIP at 5 hours in sporulation medium - replicate 3

first: Andreas

last: Hochwagen

address: 1009 Silver Center 100 Washington Square East

institute: New York University

description: Spo11-18myc ChIP in cells incubated for 5 hrs in sporulation medium

hyb_protocol: Samples were co-hybridized to custom Agilent arrays in 1X Agilent hybridization buffer at 65 degrees for 16 hours. Samples were washed according to manufacturer's instructions.

scan_protocol: Agilent scanner (default settings), 10uM resolution

data_processing: Cy3 and Cy5 levels were calculated using Agilent Feature Extractor CGH software. Background normalization, log2 ratios for each experiment and scale normalizations across each set of duplicated experiments were calculated using the sma package in R (Yang, et al., 2001), a computer language and environment for statistical computing (v2.1.0, http://www.r-project.org).

source_name: Spo11-18myc chromatin IP from cells incubated for 5 hours in sporulation medium

label_protocol: Half of the immunoprecipitated material was labeled with Cy3-dUTP (GE Healthcare) by random priming using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).

growth_protocol: Cells were incubated for 5 hours in sporulation medium before samples were isolated.

extract_protocol: 20 ml of cells were harvested after 5 hours in sporulation media. Spo11 chromatin immunoprecipitation was performed as described (Aparicio, et al., 1997), except without formaldehyde crosslinking. One tenth of the lysate was removed as an input sample. Spo11-myc was immunoprecipitated using 2 µg 9E11 (Abcam) for 16 hours at 4°C.

tag: YAH2487 (MATa/α, ho::LYS2/ho::LYS2, lys2/lys2, ura3/ura3, leu2::hisG/leu2::hisG, his3::hisG/his3::hisG, rad50S::URA3/rad50S::URA3, SPO11-18MYC::TRP1/SPO11-18MYC::TRP1)

source_name: Spo11-18myc input DNA from cells incubated for 5 hours in sporulation medium

label_protocol: One tenth of the input material was labeled with Cy5-dUTP (GE Healthcare) by random priming using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).


title: 2426 soybean nodules 13 dpi  rep_4


title: Tumour 232

city: MAJADAHONDA

first: Javier

last: Alonso

state: MADRID

address: CTRA. MAJADAHONDA-POZUELO KM 2

department: AREA DE GENÉTICA HUMANA

country: Spain

institute: INSTITUTO DE SALUD CARLOS III - IIER

description: GSM205916 and GSM207907 represent Primary Ewing Sarcoma Tumor # 17

hyb_protocol: Supplied for the manufacturer

scan_protocol: Arrays scanned in an Axon GenePix 400 scanner

data_processing: Cyclic loess method was used to scale microarrays

source_name: primary Ewing tumour

label_protocol: Supplied for the manufacturer

extract_protocol: Total RNA was extracted from frozen tumor samples using TRI-REAGENT. Quality of the RNA was assayed using the Agilent Bioanalyzer

tag: age at diagnosis:13.3y

tag: primary site:rib

tag: stage at diagnosis:localized


title: Tumour 263

description: GSM205917 and GSM207908 represent Primary Ewing Sarcoma Tumor # 18

tag: age at diagnosis:11.9y

tag: primary site:pelvis


title: Tumour 220

description: GSM205915 and GSM207906 represent Primary Ewing Sarcoma Tumor # 16

tag: age at diagnosis:12.6y

tag: primary site:femur


title: Whole genome recruitment of Rpb3 in S.cerevesiae

data_processing: Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol for array CGH that showed high correlation (r2=0.9) of bound probes between data of the two biological replicates.  Identification of bound probes and genes was made using Agilent ChIP analytics software (version 1.3) by using Whitehead Institute Per-array Neighbourhood model v1.0.  In the neighbourhood model, if candidate bound probe set p-value was less than 0.05, the central probe was marked as potentially bound, and the probe sets were required to pass one of two additional filters: the center probe in the probe set has a single probe p-value less than 0.05 or one of the flanking probes has a single point p-value less than 0.25.  These two filters cover situations where a binding event occurs midway between two probes and each detects the event, weakly.

extract_protocol: TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25 C till log phase (O. D. 600 ~ 0.8).  Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes.  Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche).  Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer.  Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp.  Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation.  Two hundred microliters of whole-cell extract was incubated with Rabbit IgG agarose beads (Sigma) for 3 hrs at 4°C with agitation.  Beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of lysis buffer plus 500 mM NaCl, twice with 1 ml of wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA) and once with 1 ml of Tris-EDTA (TE; 10 mM Tris-HCl [pH 8.0], 1 mM EDTA).  Bound material was eluted from the beads by resuspending beads in 100 µl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and incubating 10 min at 65°C with occasional agitation.  Samples were centrifuged briefly and the cross-linking was reversed by incubating overnight at 65°C.  Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 30 µl of TE.  These samples were further purified with Qiagen PCR product purification Kit.

tag: Medium

extract_protocol: TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25 C till log phase (O. D. 600 ~ 0.8).  Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes.  Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche).  Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer.  Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp.  Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. 50 ul of WCE was mixed with 150 µl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and cross-linking was reversed by incubating overnight at 65°C.  Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 50 µl of TE.  These samples were further purified with Qiagen PCR product purification Kit.


title: ssDNA enrichment in a spo11Y135F strain after 3 hours in sporulation medium - replicate 2

description: ssDNA enrichment in spo11Y135F cells at 3 hrs in sporulation medium

source_name: ssDNA in a spo11Y135F strain after 3 hours in sporulation medium

label_protocol: 1.5 μg of ssDNA was labeled with Cy5-dUTP (GE Healthcare) by random priming without denaturation using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).

growth_protocol: Cells were incubated for 3 hours in sporulation medium before samples were isolated.

extract_protocol: To isolate ssDNA, ~109 cells were fixed in 70% ethanol at –20°C. Cells were spheroplasted in sorbitol buffer (1M sorbitol, 1% beta-mercaptoethanol, 0.2 mg/ml zymolyase 100T, 0.1M EDTA, pH 7.4), lysed in NDS (0.6% SDS, 300mM EDTA, 10mM Tris.HCl, pH 9.5). DNA deproteinated with proteinase K (0.25mg/ml; Roche) at 50°C, twice phenol extracted, treated with RNase A, and stored in TE (1mM EDTA, 10mM Tris.HCl, pH 7.5) at 4°C. 25 μg DNA were digested to completion with EcoRI (New England Biolabs). ssDNA was enriched by batch absorption to BND cellulose. Briefly, a 50% slurry of BND-cellulose was equilibrated in NET (1M NaCl, 10mM Tris, pH 7.6, 1mM EDTA). Digested DNA was adjusted to 1M NaCl and ssDNA was incubated with a 500 μl bed volume of BND-cellulose for 5 minutes. Resin was washed with five bed volumes of NET. ssDNA was eluted five times with 600 μl NET + 1.8% caffeine. DNA was precipitated and concentrated for microarray analysis.

tag: A10914 (MATa/α, ho::LYS2/ho::LYS2, lys2/lys2, ura3/ura3, leu2::hisG/leu2::hisG, arg4-nsp/arg4-bgl, his4X-LEU2-URA3/his4B-LEU2, spo11-Y135F-HA::URA3/ spo11-Y135F-HA::URA3)

source_name: ssDNA in a spo11Y135F strain at 0 hours in sporulation medium

label_protocol: 1.5 μg of ssDNA was labeled with Cy3-dUTP (GE Healthcare) by random priming without denaturation using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).

growth_protocol: Samples were isolated at the time that cells were inoculated into sporulation medium (0 hrs).


title: Spo11-18myc ChIP at 5 hours in sporulation medium - replicate 2


title: Monocyte_HCMV_LY-1

description: HCMV-infected monocyte pretreated with LY, biological rep1


title: Tumour 171

description: GSM205912 and GSM207902 represent Primary Ewing Sarcoma Tumor # 12

tag: primary site:soft tissue

tag: stage at diagnosis:metastatic


title: Tumour 216

description: GSM205914 and GSM207904 represent Recurrent Ewing tumour Tumor # 14

source_name: recurrent Ewing tumour

tag: age at diagnosis:na

tag: primary site:na

tag: stage at diagnosis:na


title: Spo11-18myc ChIP at 5 hours in sporulation medium - replicate 1

label_protocol: Half of the immunoprecipitated material was labeled with Cy5-dUTP (GE Healthcare) by random priming using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).

tag: YAH2487	(MATa/α, ho::LYS2/ho::LYS2, lys2/lys2, ura3/ura3, leu2::hisG/leu2::hisG, his3::hisG/his3::hisG, rad50S::URA3/rad50S::URA3, SPO11-18MYC::TRP1/SPO11-18MYC::TRP1)

label_protocol: One tenth of the input material was labeled with Cy3-dUTP (GE Healthcare) by random priming using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).


title: Monocyte_HCMV_B11-2

description: HCMV-infected monocyte pretreated with Bay11, biological rep2

source_name: HCMV-infected monocyte pretreated with Bay11 at 4 hours post infection

tag: Tissue

tag: Virus Strain


title: 2426 soybean nodules 21 dpi  rep_1

source_name: Nodules soybean  21 dpi infected with B. japonicum regR mutant strain

tag: Genotype

tag: Growth condition

tag: Time


title: 2426 soybean nodules 21 dpi  rep_4


title: Monocyte_HCMV_LY-2

description: HCMV-infected monocyte pretreated with LY, biological rep2


title: ssDNA enrichment in a spo11Y135F strain after 3 hours in sporulation medium - replicate 1

extract_protocol: EDTA, 10mM Tris.HCl, pH 7.5) at 4°C. 25 μg DNA were digested to completion with EcoRI (New England Biolabs). ssDNA was enriched by batch absorption to BND cellulose. Briefly, a 50% slurry of BND-cellulose was equilibrated in NET (1M NaCl, 10mM Tris, pH 7.6, 1mM EDTA). Digested DNA was adjusted to 1M NaCl and ssDNA was incubated with a 500 μl bed volume of BND-cellulose for 5 minutes. Resin was washed with five bed volumes of NET. ssDNA was eluted five times with 600 μl NET + 1.8% caffeine. DNA was precipitated and concentrated for microarray analysis.


title: Monocyte_HCMV_B11-3

description: HCMV-infected monocyte pretreated with Bay11, biological rep3


title: 2426 soybean nodules 21 dpi  rep_3


title: Tumour 133

description: GSM205911 and GSM207901 represent Primary Ewing Sarcoma Tumor # 10

tag: age at diagnosis:15.2y

tag: stage at diagnosis:localized


title: Monocyte_HCMV_B11-1

description: HCMV-infected monocyte pretreated with Bay11, biological rep1


title: 2426 aerobic PSY rep_8

description: B. japonicum regR mutant strain grown aerobically

value: aerobic


title: ssDNA enrichment in a wild-type strain after 3 hours in sporulation medium - replicate 2

description: ssDNA enrichment in wild-type cells at 3 hrs in sporulation medium

scan_protocol: Agilent scanner (default settings), 5uM resolution

source_name: ssDNA in a wild-type strain after 3 hours in sporulation medium

tag: NKY1551 wild-type SK1 (MATa/α, ho::LYS2/ho::LYS2, lys2/lys2, ura3/ura3, leu2::hisG/leu2::hisG, arg4-nsp/arg4-bgl, his4X-LEU2-URA3/his4B-LEU2)

source_name: ssDNA in a wild-type strain at 0 hours in sporulation medium


title: Monocyte_HCMV-5

description: HCMV-infected monocyte, biological rep5

source_name: HCMV-infected monocytes at 4 hours post infection


title: Tumour 192

description: GSM205913 and GSM207903 represent Primary Ewing Sarcoma Tumor # 13

tag: age at diagnosis:11.0y

tag: primary site:radius


title: Tumour 127

description: GSM205910 and GSM207900 represent Recurrent Ewing tumour Tumor # 9

tag: age at diagnosis:2.9y

tag: primary site:vertebra


title: Tumour 125

description: GSM205909 and GSM207899 represent Primary Ewing Sarcoma Tumor # 8

tag: age at diagnosis:16.2y

tag: primary site:clavicle


title: 2426 aerobic PSY rep_7

source_name: B. japonicum regR mutant strain grown aerobically

tag: Medium


title: 2426 soybean nodules 21 dpi  rep_2

description: Nodules soybean  21 dpi infected with B. japonicum regR mutant strain


title: Tumour 118

description: GSM205908 and GSM207898 represent Primary Ewing Sarcoma Tumor # 7

tag: age at diagnosis:16.0y

tag: stage at diagnosis:metastatic


title: ssDNA enrichment in a dmc1Δ zip1Δ strain after 5 hours in sporulation medium - replicate 2

description: ssDNA enrichment in dmc1Δ zip1Δ cells at 5 hrs in sporulation medium

source_name: ssDNA in a dmc1Δ zip1Δ strain after 5 hours in sporulation medium

tag: Strain YAH2650 (dmc1Δ/dmc1Δ, zip1Δ/zip1Δ)

source_name: ssDNA in a dmc1Δ zip1Δ strain at 0 hours in sporulation medium


title: ssDNA enrichment in a dmc1Δ zip1Δ strain after 5 hours in sporulation medium - replicate 1


title: ssDNA enrichment in a wild-type strain after 3 hours in sporulation medium - replicate 1


title: ssDNA enrichment in a dmc1Δ ndj1Δ strain after 5 hours in sporulation medium - replicate 2

description: ssDNA enrichment in dmc1Δ ndj1Δ cells at 5 hrs in sporulation medium

source_name: ssDNA in a dmc1Δ ndj1Δ strain after 5 hours in sporulation medium

tag: Strain YAH2626 (dmc1Δ/dmc1Δ, ndj1Δ/ndj1Δ)

source_name: ssDNA in a dmc1Δ ndj1Δ strain at 0 hours in sporulation medium


title: Monocyte_HCMV-6

description: HCMV-infected monocyte, biological rep6

tag: Tissue

tag: Virus Strain


title: Monocyte_HCMV-4

description: HCMV-infected monocyte, biological rep4


title: Monocyte_HCMV-3

description: HCMV-infected monocyte, biological rep3


title: 2426 aerobic PSY rep_6

tag: Genotype

tag: Growth condition


title: ssDNA enrichment in a dmc1Δ strain with bisected chromosome XV after 5 hours in sporulation medium - replicate 2

description: ssDNA enrichment in dmc1Δ cells with bisected chromosome XV at 5 hrs in sporulation medium

source_name: ssDNA in a dmc1Δ strain with bisected chromosome XV after 5 hours in sporulation mediumssDNA in a dmc1Δ strain with bisected chromosome XV after 5 hours in sporulation medium

source_name: ssDNA in a dmc1Δ strain with bisected chromosome XV at 0 hours in sporulation medium


title: 2426 aerobic PSY rep_5


title: 2426 aerobic PSY rep_4


title: Lin-52-smoothed

status: Public on Nov 14 2007

city: Berkeley

first: Soyeon

last: Ahn

state: CA

address: University of California, Berkeley

department: Statistics

institute: UC Berkeley

description: normalized and smoothed log ratio data (ChIP/input DNA)

hyb_protocol: Samples were prepared and hybridized to GeneChip Drosophila Tiling 1.0R Array according to Affymetrix specifications.

scan_protocol: Affymetrix

data_processing:
Identification of MMB-enriched regions: The entire dataset was analyzed with the R software. After background correction, raw data were quantile-normalized using GCRMA (Bolstad et al. 2003; Wu et al. 2004). Enrichment was quantified by first computing a difference of average log-intensities of ChIP and control DNA samples followed by the smoothing of the data over a moving window of 500 bp. These windowlevel statistics were used to calculate the p-value by a nonparametric procedure as previously described except that a simple mean rather than a trimmed mean was used (Schwartz et al. 2006).
Defining enriched regions and peaks: In order to find MMB-enriched regions, a nonparametric p-value for the smoothed, window-level scores was computed and adjusted for multiple testing to control false discovery rate (FDR), by the Storey q-value method (Storey and Tibshirani 2003). A region was defined as potentially enriched if it contained one or more probes with q-value below 0.01. If a region contained less than 5 probes, it was discarded. If the next probe with q-value below 0.01 was within 1 kb of an enriched region, it was declared to belong to the same enriched region.
Peaks representing the putative MMB complex binding sites were then defined, based on the following criteria. First, if region's length was less than 2 kb, then we took the local maximum of the region as a peak. Second, when a region's length was bigger than 2 kb, we considered the local maximum among +/- 15 neighboring probes (approximately 500 bp), with a 1 kb minimum gap between peaks. Third, since we considered the joined enriched regions that might contain non-enriched regions for some protein, we also filtered out peaks when the log2 ratio was less than 0.5 or the q-value was above 0.03, to remove peaks which might not be real.

label: Biotin

organism: Drosophila melanogaster

source_name: ChIP with antibody against Lin-52

growth_protocol: Drosophila Kc cells were grown in suspension at 25°C in Schneider’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and   Antibiotic-Antimycotic solution (Invitrogen).

extract_protocol: We performed chromatin immunoprecipitations (ChIP) using antibodies against several MMB members (Myb, Mip120, Mip130, E2F2 and Lin-52)

tag: Drosophila Kc cells

source_name: Input control DNA


title: Mip120-smoothed

source_name: ChIP with antibody against Mip120

label_protocol: Samples were prepared and hybridized to GeneChip Drosophila Tiling 1.0R Array according to Affymetrix specifications.


title: E2F2-smoothed

source_name: ChIP with antibody against E2F2

growth_protocol: Drosophila Kc cells were grown in suspension at 25°C in Schneider’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and Antibiotic-Antimycotic solution (Invitrogen).


title: Mip130-smoothed

source_name: ChIP with antibody against Mip130


title: ssDNA enrichment in a dmc1Δ ndj1Δ strain after 5 hours in sporulation medium - replicate 1


title: Monocyte_HCMV-1

description: HCMV-infected monocyte, biological rep1


title: ssDNA enrichment in a dmc1Δ strain with bisected chromosome XV after 5 hours in sporulation medium - replicate 1

source_name: ssDNA in a dmc1Δ strain with bisected chromosome XV after 5 hours in sporulation medium


title: 2426 aerobic PSY rep_3


title: Tumour 50

description: GSM205905 and GSM207893 represent Primary Ewing Sarcoma Tumor # 2

source_name: primary Ewing tumour

tag: age at diagnosis:12.2y

tag: primary site:femur

tag: stage at diagnosis:localized


title: Myb-smoothed

source_name: ChIP with antibody against Myb


title: 2426 aerobic PSY rep_2


title: Tumour 117

description: GSM205907 and GSM207897 represent Primary Ewing Sarcoma Tumor # 6

tag: age at diagnosis:13.7y

tag: primary site:rib


title: Tumour 33

description: GSM205904 and GSM207892 represent Primary Ewing Sarcoma Tumor # 1

tag: age at diagnosis:3.3y

tag: primary site:soft tissue


title: Tumour 99

description: GSM205906 and GSM207894 represent Primary Ewing Sarcoma Tumor # 3

tag: age at diagnosis:14.7y

tag: primary site:vertebra


title: Monocyte_HCMV-2

description: HCMV-infected monocyte, biological rep2


title: 2426 aerobic PSY rep_1

tag: Medium


title: ssDNA enrichment in a dmc1Δ strain after 5 hours in sporulation medium - replicate 2

description: ssDNA enrichment in dmc1Δ cells at 5 hrs in sporulation medium

data_processing: Cy3 and Cy5 levels were calculated using Agilent Feature Extractor CGH software.  Background normalization, log2 ratios for each experiment and scale normalizations across each set of duplicated experiments were calculated using the sma package in R (Yang, et al., 2001), a computer language and environment for statistical computing (v2.1.0, http://www.r-project.org).

source_name: ssDNA in a dmc1Δ strain after 5 hours in sporulation medium

label_protocol: 1.5 μg of ssDNA was labeled with Cy5-dUTP (GE Healthcare) by random priming without denaturation using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs).  Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).

tag: Strain NKY1455 (dmc1Δ/dmc1Δ)

source_name: ssDNA enrichment in a dmc1Δ strain at 0 hours in sporulation medium

label_protocol: 1.5 μg of ssDNA was labeled with Cy3-dUTP (GE Healthcare) by random priming without denaturation using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs).  Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore).


title: ssDNA enrichment in a dmc1Δ strain after 5 hours in sporulation medium - replicate 1

tag: Strain NKY1455 (dmc1Δ)


title: Universal Mix Replicate 1

city: Tokyo

first: Fuyuki

last: Miya

state: Tokyo

address: 35 Shinanomachi, Shinjuku-ku

department: School of Medicine

country: Japan

institute: Keio University

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to universal tissue mix.

hyb_protocol: Hybridized for 20 h at 50 °C. Hybridization buffer and washing protocol was followed by the protocol supplied by Hitachi Software Engineering Co., Ltd. (DNA Chip Research Inc. / AceGene website)

scan_protocol: GenePix4100A (Axon) was used for scanning. Images were quantified using GenePixPro Software (version 4.1, Axon).

data_processing: Log2-ratios of the median subtracted background intensity levels were analyzed. Data from each microarray were normalized by LOWESS normalization using R software.

source_name: Universal tissue mix total RNA

label_protocol: Amino Allyl aRNA was synthesized by Amino Allyl MessageAmp™ aRNA Amplification Kit (Ambion). CyeDye coupling and fragmentation were performed using the protocol supplied by Hitachi Software Engineering Co., Ltd. (DNA Chip Research Inc. / AceGene website)

extract_protocol: Purchased total RNA was purified further with an RNeasy Mini Kit (QIAGEN) and RNase-free DNase Set (QIAGEN).

tag: Mixture of RNA with the equal amount from 24 tissues aRNA

source_name: Universal Tissue Mix

tag: Mixture of RNA with the equal amount from each tissues aRNA


title: AGD lesion fish 2 (IF3L) vs AGD no lesion fish 2 (IF3NL)

source_name: Total RNA from AGD-affected gill lesion tissue at 36 days post inoculation with Neoparamoeba perurans

label_protocol:
2μg of total RNA from each fish was subjected to 1 round of amplification by using the Amino Allyl MessageAmp I aRNA amplification kit (Ambion)
Coupling of aRNA was performed using either Cy3 or Cy5 dye according to the Amino Ally MessageAMP™ I aRNA Amplification Kit (Ambion) protocol with minor modifications. Briefly, one vial of Cy3 or Cy5 reactive dyes (Amersham) was resuspended in 22µl of DMSO prior to coupling. 3µg of aRNA was dried to completion in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY).  The aRNA was resuspended in 4.5µl of coupling buffer. Thereafter, 5.5µl of dye was added to the aRNA:coupling buffer and incubated for 30 minutes in the dark. 2.25µl of 4M hydroxylalamine was then added to quench the reaction followed by 12.75µl of water to obtain a total volume to 30µl. Dye-labeled aRNA purification was performed according to the manufacturer’s instructions.

tag: AGD-affected fish 2, AGD lesion tissue, IF3L

tag: Dissected lesion tissue from AGD-affected gill arch

tag: Fish inoculated with Neoparamoeba perurans, the aetiological agent of AGD

tag: weight

source_name: Total RNA from AGD-affected gill non-lesion tissue at 36 days post inoculation with Neoparamoeba perurans

tag: AGD-affected fish 2, AGD no lesion tissue, IF3NL

tag: Dissected non-lesion tissue from AGD-affected gill arch

tag: Weight


title: Whole Brain Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to whole brain.

source_name: Whole brain total RNA

tag: Pooled Human whole brain total RNA


title: Whole Brain Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to whole brain.


title: M. tuberculosis CDC1551 24hr phagosome expression profile

city: Ithaca

first: Kyle

middle: Herman

last: Rohde

address: Vet. Med. Center, C5 101

department: Microbiology and Immunology

institute: Cornell University

description: nothing to add

hyb_protocol: 5-10 ug of Cy-labeled Alexa-labeled aRNA from paired samples was dried using a Speedvac and resuspended in 50 l of hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 25 g salmon sperm DNA).  Samples were denatured at 95oC for 5 min and briefly cooled to 60oC before being applied to arrays under a glass LifterSlip (Erie Scientific).  Slides were prehybridized for 1 hr in 25% formamide, 5X SSC, 0.1% SDS, 1% BSA and washed with H2O and isopropanol.  Labeled targets were hybridized to microarrays in humidified slide chambers (Corning) at 45oC for 16-18 hr.  Arrays were washed sequentially with buffer 1 (2X SSC, 0.1% SDS) pre-warmed to 45oC, buffer 2 (0.2X SSC, 0.1% SDS), buffer 3 (0.2X SSC), and buffer 4 (0.05X SSC).  Slides were briefly dipped in de-ionized ultrafiltered water before being dried by centrifugation (700 X g, 10 min).

scan_protocol: Microarrays were scanned with a GenePix 4000B instrument (Axon Instruments, Inc.) with preliminary image analysis, spot intensity determination, background measurements, spot quality assessment and flagging conducted using Imagene software (version 6.0, Biodiscovery).  PMT settings were adjusted to equalize the two channels based on equal intensity of positive control spots.  PMT were also adjusted such that only a few spots were saturated to maximize detection of weak spots and maintain most intensities with linear range.

data_processing: Poor quality spots with signal intensities less than three standard deviations above background were excluded from further analysis.  Subsequent normalization, statistical analysis, and visualization of array data were performed with Genespring 7.3 (Agilent).

label: Alexa 647

organism: Mycobacterium tuberculosis

source_name: Mycobacterium tuberculosis, untreated macrophage, 24 hr

label_protocol: Using the MessageAmpTM-II Bacteria RNA Amplification system (Ambion), we amplified 100-250 ng total RNA to generate sufficient RNA from mycobacteria isolated from infected MØ for microarray hybrization.  Briefly, total bacterial RNA was first polyadenylated by E. coli poly(A) polymerase.  Using an oligo(dT) primer that incorporated a T7 promoter, poly(A) tailed RNA was reverse transcribed to yield double-stranded cDNA.  The resulting cDNA served as the template for in vitro transcription (IVT) by T7 RNA polymerase which generates multiple antisense RNA (aRNA) copies of each transcript in the sample.  Amino-allyl UTP was incorporated into aRNA during the IVT reaction to permit fluorophore post-labeling.  According the manufacturer recommendation for consistent amplification results, a standardized RNA isolation protocol was used to generate all template RNA and master mixes of reaction components were prepared for each step.  In addition, care was taken to use the same amount of starting template and identical incubation times and temperatures for all samples to be compared.    amino-allyl modified aRNA were labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen) and purified using a MegaClear kit (Ambion).

growth_protocol: The clinical isolate strain CDC1551 of M. tuberculosis (MTb) and the attenuated vaccine strain M. bovis BCG Pasteur (BCG) were routinely cultured in 7H9-OADC medium without shaking in 75 cm2 vented tissue culture flasks (T-75).  Bone marrow-derived macrophages (MØ) were isolated from C57BL/6 mice and grown in DMEM supplemented with 10% FCS, 20% L cell-conditioned medium and antibiotics (penicillin and streptomycin).   Macrophage media lacking antibiotics was added at least 24 hr before initiation of mycobacterial infections.

extract_protocol: Addition of a lysis buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate, and 0.1M -mercaptoethanol) selectively lysed MØ, halted RNA transcription/degradation while leaving mycobacteria intact as previously described (Butcher et al., 1998; Mangan et al., 2002).  Samples were vortexed and passed five times through a 21 gauge needle to shear MØ and reduce viscosity.  Intracellular mycobacteria were recovered by centrifugation at 3500 rpm for 30min.   Pelleted BCG or MTb were digested with 5g/ml lysozyme before being lysed in 650C Trizol using a BeadBeater and 0.1mm silicon beads.  Total RNA was isolated from Trizol lysates by chloroform extraction followed by addition of ethanol and direct application to Qiagen RNeasy column purification.  Finally, residual DNA contamination was removed using Turbo DNAfree DNase (Ambion).

treatment_protocol: Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4).  MØ were infected (MOI ~20:1) with Mtb or BCG grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks.  Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated.  Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli.  For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.

tag: Mtb strain CDC1551

label: Alexa 555

source_name: Extracellular Control M. tuberculosis, 24hr in infection media, no macrophages


title: Trachea Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to trachea.

source_name: Trachea total RNA

tag: Pooled Human trachea total RNA


title: Universal Mix Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to universal tissue mix.


title: Uterus Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to uterus.

source_name: Uterus total RNA

tag: Pooled Human uterus total RNA


title: Stomach Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to stomach.

source_name: Stomach total RNA

tag: Pooled Human stomach total RNA


title: Thymus Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to thymus.

source_name: Thymus total RNA

tag: Pooled Human thymus total RNA


title: Uterus Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to uterus.


title: Thyroid Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to thyroid.

source_name: Thyroid total RNA

tag: Pooled Human thyroid total RNA


title: Testis Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to testis.

source_name: Testis total RNA

tag: Pooled Human testis total RNA


title: BCG 2hr phagosomal profile

description: nothing to add

hyb_protocol: 5-10 g of Cy-labeled cDNA or Alexa-labeled aRNA from paired samples was dried using a Speedvac and resuspended in 50 l of hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 25 g salmon sperm DNA).  Samples were denatured at 95oC for 5 min and briefly cooled to 60oC before being applied to arrays under a glass LifterSlip (Erie Scientific).  Slides were prehybridized for 1 hr in 25% formamide, 5X SSC, 0.1% SDS, 1% BSA and washed with H2O and isopropanol.  Labeled targets were hybridized to microarrays in humidified slide chambers (Corning) at 45oC for 16-18 hr.  Arrays were washed sequentially with buffer 1 (2X SSC, 0.1% SDS) pre-warmed to 45oC, buffer 2 (0.2X SSC, 0.1% SDS), buffer 3 (0.2X SSC), and buffer 4 (0.05X SSC).  Slides were briefly dipped in de-ionized ultrafiltered water before being dried by centrifugation (700 X g, 10 min).

source_name: Phagosomal BCG after 2hr macrophage infection

tag: M. bovis BCG Pasteur, grown in standing 7H9 broth cultures to mid-log phase

source_name: Extracellular Control BCG, 2hr in infection media, no macrophages


title: Trachea Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to trachea.

tag: Mixture of RNA with the equal amount from each tissues aRNA


title: Stomach Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to stomach.


title: Thyroid Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to thyroid.


title: Effect of macrophage contact (phagocytosis inhibited with cytochalasin D) on Mtb gene expression

description: Nothing to add.

hyb_protocol: 5-10 ug of Alexa-labeled aRNA from paired samples was dried using a Speedvac and resuspended in 50 ul of hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 25 g salmon sperm DNA).  Samples were denatured at 95oC for 5 min and briefly cooled to 60oC before being applied to arrays under a glass LifterSlip (Erie Scientific).  Slides were prehybridized for 1 hr in 25% formamide, 5X SSC, 0.1% SDS, 1% BSA and washed with H2O and isopropanol.  Labeled targets were hybridized to microarrays in humidified slide chambers (Corning) at 45oC for 16-18 hr.  Arrays were washed sequentially with buffer 1 (2X SSC, 0.1% SDS) pre-warmed to 45oC, buffer 2 (0.2X SSC, 0.1% SDS), buffer 3 (0.2X SSC), and buffer 4 (0.05X SSC).  Slides were briefly dipped in de-ionized ultrafiltered water before being dried by centrifugation (700 X g, 10 min).

scan_protocol: Microarrays were scanned with a GenePix 4000B instrument (Axon Instruments, Inc.) with preliminary image analysis, spot intensity determination, background measurements, spot quality assessment and flagging conducted using Imagene software (version 6.0, Biodiscovery).  PMT levels for scanning were set to equalize signal intensity of positive control spots and minimize saturated spots.

data_processing: Preliminary image analysis, spot intensity determination, background measurements, spot quality assessment and flagging conducted using Imagene software (version 6.0, Biodiscovery).  Poor quality spots with signal intensities less than three standard deviations above background were excluded from further analysis.  Subsequent normalization, statistical analysis, and visualization of array data were performed with Genespring 7.3 (Agilent).  Lowess normalization was used, Genespring Cross-Gene error model was activated.  Genes with significant changes in expression levels relative to controls were identified based on fold change (1.5-fold) and reproducibility between replicates (p-value <0.05).

source_name: Mycobacterium tuberculosis, cytochalasin D treated macrophage, 2 hr

label_protocol: Using the MessageAmpTM-II Bacteria RNA Amplification system (Ambion), we amplified 100-250 ng total RNA to generate sufficient RNA from mycobacteria isolated from infected MØ for microarray hybrization.  Briefly, total bacterial RNA was first polyadenylated by E. coli poly(A) polymerase.  Using an oligo(dT) primer that incorporated a T7 promoter, poly(A) tailed RNA was reverse transcribed to yield double-stranded cDNA.  The resulting cDNA served as the template for in vitro transcription (IVT) by T7 RNA polymerase which generates multiple antisense RNA (aRNA) copies of each transcript in the sample.  Amino-allyl UTP was incorporated into aRNA during the IVT reaction to permit fluorophore post-labeling. amino-allyl modified aRNA were labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen) and purified using a MegaClear kit (Ambion).

extract_protocol: Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4).  MØ were infected (MOI ~20:1) with Mtb or BCG grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks.  Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated.  Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli.  For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.  Addition of a lysis buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate, and 0.1M -mercaptoethanol) selectively lysed MØ, halted RNA transcription/degradation while leaving mycobacteria intact as previously described {Butcher, 1998 #139; Mangan, 2002 #140}.  Samples were vortexed and passed five times through a 21 gauge needle to shear MØ and reduce viscosity.  Intracellular mycobacteria were recovered by centrifugation at 3500 rpm for 30min.   Pelleted BCG or MTb were digested with 5 ug/ml lysozyme before being lysed in 65C Trizol using a BeadBeater and 0.1mm silicon beads.  Total RNA was isolated from Trizol lysates by chloroform extraction followed by addition of ethanol and direct application to Qiagen RNeasy column purification.  Finally, residual DNA contamination was removed using Turbo DNAfree DNase (Ambion).

treatment_protocol: Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4).  MØ were infected (MOI ~20:1) with Mtb grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks.  Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated.  Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli.  For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.  MØ were pretreated with  5 micromolar cytochalasin D (cytoD) for 20 min prior to infection to inhibit phagocytosis, respectively, and maintained throughout the infection period.  Control mycobacteria not exposed to MØ were treated with identical inhibitor concentrations.  Microscopic examination of cytochalasin D-pretreated MØ incubated with carboxyfluorescein-labeled silica beads and BCG confirmed inhibition of phagocytosis.

tag: Strain CDC1551

source_name: Mycobacterium tuberculosis, cytochalasin D treated no macrophage extracellular controls

tag: strain CDC1551


title: BCG 24hr phagosomal profile

description: nothing to add

source_name: Phagosomal BCG after 24hr macrophage infection

tag: M. bovis BCG Pasteur isolated from resting bone marrow derived macrophage phagosomes 24hr post-infection

source_name: Extracellular Control BCG, 24hr in infection media, no macrophages

tag: M. bovis BCG Pasteur "no macrophage" controls, 24hr


title: M. tuberculosis CDC1551 2hr phagosome expression profile

description: nothing to add

source_name: Mycobacterium tuberculosis, untreated macrophage, 2 hr

tag: M. tuberculosis strain CDC1551

source_name: Extracellular Control M. tuberculosis, 2hr in infection media, no macrophages


title: Mtb response to pH 6.5 (6 replicates)

description: Nothing to add.

hyb_protocol: 5-10 ug of Cy-labeled cDNA or Alexa-labeled aRNA from paired samples was dried using a Speedvac and resuspended in 50 ul of hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 25 g salmon sperm DNA).  Samples were denatured at 95oC for 5 min and briefly cooled to 60oC before being applied to arrays under a glass LifterSlip (Erie Scientific).  Slides were prehybridized for 1 hr in 25% formamide, 5X SSC, 0.1% SDS, 1% BSA and washed with H2O and isopropanol.  Labeled targets were hybridized to microarrays in humidified slide chambers (Corning) at 45oC for 16-18 hr.  Arrays were washed sequentially with buffer 1 (2X SSC, 0.1% SDS) pre-warmed to 45oC, buffer 2 (0.2X SSC, 0.1% SDS), buffer 3 (0.2X SSC), and buffer 4 (0.05X SSC).  Slides were briefly dipped in de-ionized ultrafiltered water before being dried by centrifugation (700 X g, 10 min).

source_name: Mycobacterium tuberculosis, pH 7.0 control, 2 hr

treatment_protocol: The transcriptional responses of Mtb CDC1551 to low pH were examined in buffered 7H9-OADC medium. The pH 5.5 and 6.5 adjusted media were buffered with 100 mM 2-(4-morpholino)-ethane sulfonic acid (MES), and the pH 7.0 adjusted medium was buffered with 100 mM 3-(N-morpholino) propanesulfonic acid (MOPS) (Piddington et al., 2000).  A 50 ml starter culture of Mtb grown in 7H9-OADC (pH 7.0) to early log phase (OD ~0.25) was split into three 15 mL aliquots, centrifuged, and the pellets were resuspended in 12mL of 7H9-OADC buffered at pH 5.5, pH 6.5 or pH 7.0.  Cells were incubated at 37 oC for 2 hr before RNA isolation, amplification and analysis by microarray as detailed below.  Mtb responses to pH were examined in three independent experiments and for each experimental replicate, technical replicates were conducted in which the amplified RNA was labeled and hybridized to microarrays a second time.  The presented microarray data are the average of six total datasets (three experimental replicates and two technical replicates).

tag: Strain CDC1551, grown in 7H9 broth standing cultures to OK ~0.25

source_name: Mycobacterium tuberculosis, pH 6.5 acid stress, 2 hr


title: Phagosomal induced Mtb genes affected by Concanamycin A inhibition of phagosome acidification

description: Nothing to add.

extract_protocol: Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4).  MØ were infected (MOI ~20:1) with Mtb or BCG grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks.  Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated.  Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli.  For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.  Addition of a lysis buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate, and 0.1M -mercaptoethanol) selectively lysed MØ, halted RNA transcription/degradation while leaving mycobacteria intact as previously described {Butcher, 1998 #139; Mangan, 2002 #140}.  Samples were vortexed and passed five times through a 21 gauge needle to shear MØ and reduce viscosity.  Intracellular mycobacteria were recovered by centrifugation at 3500 rpm for 30min.   Pelleted BCG or MTb were digested with 5 ug/ml lysozyme before being lysed in 65C Trizol using a BeadBeater and 0.1mm silicon beads.  Total RNA was isolated from Trizol lysates by chloroform extraction followed by addition of ethanol and direct application to Qiagen RNeasy column purification.  Finally, residual DNA contamination was removed using Turbo DNAfree DNase (Ambion).  Where indicated, MØ were pretreated with 100 nM concanamycin A (CcA) for 20 min prior to infection to inhibit phagosome acidification and maintained throughout the infection period.

treatment_protocol:
Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4).  MØ were infected (MOI ~20:1) with Mtb grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks.  Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated.  Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli.  For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.  MØ were pretreated with  100 nM concanamycin A (CcA) for 20 min prior to infection to inhibit phagosome acidification and maintained throughout the infection period.  Control mycobacteria not exposed to MØ were treated with identical inhibitor concentrations
Concanamycin A (100 nM) was shown to effectively block acidification of mycobacterial phagosomes using carboxyfluoroscein-modified beads and BCG as previously described

tag: Strain CDC1551, grown in 7H9 broth standing cultures to OK ~0.25.  Isolated from infected bone marrow derived murine macrophages 2hr post-infection

source_name: Mycobacterium tuberculosis, Concanamycin A treated macrophages, 2 hr

tag: Strain CDC1551, grown in 7H9 broth standing cultures to OK ~0.25.  Isolated from infected bone marrow derived murine macrophages (pretreated with 100nM concanamycin A for 20min prior to infection) 2hr post-infection


title: Spinal Code Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to spinal code.

source_name: Spinal code total RNA

tag: Pooled Human spinal code total RNA

source_name: Universal Tissue Mix


title: Small Intestine Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to small intestine.

source_name: Small intestine total RNA

tag: Pooled Human small intestine total RNA


title: Testis Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to testis.


title: Thymus Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to thymus.

source_name: Thymus total RNA


title: Spinal Code Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to spinal code.


title: Mtb response to pH 5.5 (6 replicates)

description: Nothing to add.

tag: Strain CDC1551, grown in 7H9 broth standing cultures to OD ~0.25

source_name: Mycobacterium tuberculosis, pH 5.5 acid stress, 2 hr


title: Spleen Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to spleen.

source_name: Spleen total RNA

tag: Pooled Human spleen total RNA


title: Spleen Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to spleen.


title: Small Intestine Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to small intestine.


title: Skeletal Muscle Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to skeletal muscle.

source_name: Skeletal muscle total RNA

tag: Pooled Human skeletal muscle total RNA


title: MCM-IP_aF_orc6-td-establishment-dyeswap

city: Cambridge

first: Stephen

middle: P

last: Bell

state: MA

address: 68-630, 77 Massachusetts Ave.

institute: MIT

description: ORC CHIP in G2/M

hyb_protocol: standard Agilent hybridization and wash protocols

scan_protocol: Agilent Scanner

data_processing: For each cohybridization, Cy3 and Cy5 levels were calculated using Agilent Feature Extractor CGH software.  Log2 ratios for each experiment and scale normalizations across each set of duplicated experiments were calculated using the sma package (Yang et al. 2002) in R, a computer language and environment for statistical computing (v2.1.0, http://www.r-project.org).

label_protocol: Half of the immunoprecipitated DNA and 5 μg of ChIP input DNA were differentially labeled with 2 nmol of either Cy3-dUTP or Cy5-dUTP (GE Healthcare) using 4 μg random nonomer oligo (IDT) and 0.5 μl 50,000 u/ml Klenowexo- (NEB) in 40 μl. IP and input samples were mixed after labeling and unincorporated dyes were removed using a microcon filter (Millipore YM-30) by washing the samples three times with 0.4 ml TE.

extract_protocol: ChIP assays were performed as described previously (Aparicio et al., 1997).  Thirty-three ml of culture was collected for each ChIP sample.  One third of the resulting cell lysate was used for Mcm2-7 IP using an anti-Mcm2-7 polyclonal antibody (UM185), one-third for ORC IP using an anti-ORC polyclonal antibody (1108).

tag: ChIP IP with anti-MCM antibody. Orc6-td cells released from nocodazole into aF at 37-deg in YP-gal.

source_name: input

label_protocol: see above

extract_protocol: see above

tag: ChIP input


title: Skeletal Muscle Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to skeletal muscle.


title: Salivary Gland Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to salivary gland.

source_name: Salivary gland total RNA

tag: Pooled Human salivary gland total RNA


title: Prostate Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to prostate.

source_name: Prostate total RNA

tag: Pooled Human prostate total RNA

tag: Mixture of RNA with the equal amount from each tissues aRNA


title: Placenta Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to placenta.

source_name: Placenta total RNA

tag: Pooled Human placenta total RNA


title: MCM-IP_aF_orc6-td-maintenance-dyespwap

description: ORC CHIP in G2/M

tag: ChIP IP with anti-MCM antibody. Orc6-td cells arrested in aF at 25 degrees and then shifted to 37-deg in YP-gal.


title: MCM-IP_aF_wild-type-maintenance

description: ORC CHIP in G2/M

tag: ChIP IP with anti-MCM antibody. Cells arrested in aF at 25 degrees and then shifted to 37-deg in YP-gal.

tag: ChIP input.


title: Lung Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to lung.

source_name: Lung total RNA

tag: Pooled Human lung total RNA


title: MCM-IP_aF_orc6-td-establishment

description: ORC CHIP in G2/M


title: Lung Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to lung.


title: MCM-IP_aF_orc6-td-maintenance

description: ORC CHIP in G2/M


title: Prostate Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to prostate.


title: Placenta Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to placenta.


title: ORC-IP_aF_orc6_td-establishment

description: ORC CHIP in G2/M

tag: ChIP IP with anti-ORC antibody.  Orc6-td cells released from nocodazole into aF at restrictive conditions.

tag: ChIP input DNA


title: Liver Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to liver.

source_name: Liver total RNA

tag: Pooled Human liver total RNA


title: MCM-IP_aF_wild-type-establishment

description: ORC CHIP in G2/M

tag: ChIP IP with anti-MCM antibody.  Cells released from nocodazole into aF at 37-deg in YP-gal.


title: ORC-IP_nocodazole_orc6_td

description: ORC CHIP in G2/M

tag: ChIP IP with anti-ORC antibody in orc6-td strain in nocodazole


title: Kidney Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to kidney.

source_name: Kidney total RNA

tag: Pooled Human kidney total RNA


title: Salivary Gland Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to salivary gland.


title: ORC-IP_nocodazole_wild-type

description: ORC CHIP in G2/M

tag: ChIP IP with anti-ORC antibody


title: MCM-IP_aF_wild-type-establishment-dyeswap

description: ORC CHIP in G2/M

tag: ChIP IP with anti-MCM antibody. Cells released from nocodazole into aF at 37-deg in YP-gal.


title: MCM-IP_aF_wild-type-maintenance-dyeswap

description: ORC CHIP in G2/M

tag: ChIP input


title: Heart Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to heart.

source_name: Heart total RNA

tag: Pooled Human heart total RNA


title: Heart Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to heart.


title: ORC-IP_nocodazole_wild-type-dyeswap

description: ORC CHIP in G2/M

tag: ChIP IP with anti-ORC antibody.  Cells arrested in nocodazole at 25-deg.


title: L(3)MBT knock-down 2

hyb_protocol: Following fragmentation, 15 microg of cRNA were hybridized to Drosophila Genechips (Drosgenome 1) according to Affymetrix specifications.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.

data_processing: Fluorescence intensities of scanned arrays were analyzed with Affymetrix GeneChip Operating Software (GCOS v1.2.0). CEL output files were background corrected and quantile normalized. Statistical analysis, data processing, and visualization were performed using the R and Bioconductor libraries.

source_name: Drosophila Kc cells

label_protocol: Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 10 microg total RNA.

extract_protocol: Total RNA was extracted from RNAi-transfected cells after 4 days using RNeasy Mini Kit (Qiagen).

treatment_protocol:
Control : RNAi in Drosophila Kc cells with a nonspecific RNA derived from a pBSK+ (named SK+).
Treatment : RNAi to deplete Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and RBF2) and L(3)MBT.

tag: Strain

value: Drosophila Kc cells/

tag: Karyotype

value: tetraploid female karyotype/

tag: Treatment

value: 4 days RNAi to deplete L(3)MBT


title: Liver Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to liver.

tag: Mixture of RNA with the equal amount from each tissues aRNA


title: ORC-IP_nocodazole_orc6_td-dyeswap

description: ORC CHIP in G2/M


title: Fetal Liver Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to fetal liver.

source_name: Fetal liver total RNA

tag: Pooled Human fetal liver total RNA


title: L(3)MBT knock-down 1


title: Kidney Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to kidney.


title: E2F1 knock-down 2

value: 4 days RNAi to deplete E2F1


title: Frontal Lobe Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to frontal lobe.

source_name: Fetal lobe total RNA

tag: Pooled Human frontal lobe total RNA


title: Colon Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to colon.

source_name: Colon total RNA

tag: Pooled Human colon total RNA

source_name: Universal Tissue Mix


title: E2F1 knock-down 1


title: Fetal Liver Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to fetal liver.


title: E2F1 knock-down 3


title: Frontal Lobe Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to frontal lobe.


title: Colon Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to colon.


title: Fetal Brain Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to fetal brain.

source_name: Fetal brain total RNA

tag: Pooled Human fetal brain total RNA


title: Mip40 knock-down 3

value: 4 days RNAi to deplete Mip40


title: Mip40 knock-down 1


title: Mip40 knock-down 2


title: Bone Marrow Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to bone marrow.

source_name: Bone marrow total RNA

tag: Pooled Human bone marrow total RNA


title: Cerebellum Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to cerebellum.

source_name: Cerebellum total RNA

tag: Pooled Human cerebellum total RNA


title: Cerebellum Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to cerebellum.


title: Lin-52 knock-down 3

value: 4 days RNAi to deplete Lin-52


title: Fetal Brain Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to fetal brain.


title: E2F2 knock-down 3

value: 4 days RNAi to deplete E2F2


title: Bone Marrow Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to bone marrow.


title: Adrenal Gland Replicate 2

description: Dye-swap replicate 2 of 2. Comparizon with universal tissue mix to adrenal gland.

source_name: Adrenal gland total RNA

tag: Pooled Human adrenal gland total RNA


title: Lin-52 knock-down 2


title: Adrenal Gland Replicate 1

description: Dye-swap replicate 1 of 2. Comparizon with universal tissue mix to adrenal gland.


title: RBFs (RBF1 and RBF2) knock-down 1

extract_protocol: Total RNA was extracted from RNAi-transfected cells after 4 days using RNeasy Mini Kit (Qiagen)

value: 4 days RNAi to deplete both RBF1 and RBF2


title: L(3)MBT knock-down 3


title: E2F2 knock-down 2


title: RBFs (RBF1 and RBF2) knock-down 2


title: Lin-52 knock-down 1


title: Mip130 knock-down 1

value: 4 days RNAi to deplete Mip130


title: Myb knock-down 2

value: 4 days RNAi to deplete Myb


title: SmutansUA159_Mupricin_rep4

city: Gainesville

first: Robert

middle: A

last: Burne

state: FL

address: 1600 SW Archer Rd. BOX 100424

department: Oral Biology

institute: University of Florida

description: Streptococcus mutans wildtype, mupirocin treated

hyb_protocol: Hybridizations were carried out in a Maui 4-chamber hybridization system (BioMicro Systems, Salt Lake City, Utah) for 17 h at 42ºC. The slides were then washed according to TIGR protocols (SOT# M008, version 2.1, 11/06).

scan_protocol: The slides were scanned using a GenePix Scanner (Axon Instruments Inc., Union City, CA).

data_processing: After the slides were scanned, single-channel images were loaded into TIGR Spotfinder software and overlayed. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, then the intensity values of each spot were measured. Data was normalized using Microarray data analysis software (MIDAS), by using LOWESS and Iterative Log Mean Centering with default settings.

organism: Streptococcus mutans

source_name: S. mutans UA159 mup treated

label_protocol: All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy3)-dUTP

growth_protocol: Cells were grown in the chemically-defined medium FMC  to an OD600 of 0.3 in 5% CO2 at 37°C

extract_protocol: RNA from S. mutans was isolated from 50 ml cultures that were grown under the desired conditions and harvested by centrifugation at 4ºC. Pelleted cells were resuspended in 400 ml of DEPC-treated water, 800 ml of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at one minute intervals. Cells were then pelleted, resuspended in 500 ml Tris-EDTA (50:10) buffer and transferred to 1.5 ml screw cap tubes containing sterile glass beads (0.1 mm avg. diam.; Biospec, Bartlesville, OK), 100 ml of 1% SDS and 650 ml of acid phenol:chloroform (5:1). The mixture was homogenized in a Bead Beater twice for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4ºC. Two additional hot acid phenol:chloroform extractions were performed followed by one extraction with chloroform:isoamyl-alcohol (24:1). RNA was precipitated in the presence of isopropanol and sodium acetate, pH 5, at -20ºC for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in DEPC-treated water and digested with DNAseI (Ambion, Austin, TX). The RNA was re-purified and treated on-column with DNaseI using the RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA), as recommended by the supplier.

treatment_protocol: Treated with 500 ng/ml of mupirocin for 20 minutes at 37°C once OD600 of 0.3 was reached

tag: Strain UA159

source_name: S. mutans UA159 grown in BHI

label_protocol: All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy5)-dUTP

growth_protocol:
Growth Media: Brain Heart Infusion
OD600: 0.5
5% CO2, 37°C


title: Mip130 knock-down 3


title: E2F2 knock-down 1


title: Myb knock-down 3


title: Mip120 knock-down 3

value: 4 days RNAi to deplete Mip120


title: RBFs (RBF1 and RBF2) knock-down 3

hyb_protocol: Following fragmentation, 15microg of cRNA were hybridized to Drosophila Genechips (Drosgenome 1) according to Affymetrix specifications.


title: Mip130 knock-down 2


title: Mip120 knock-down 2


title: SmutansUA159_Mupricin_rep1

description: Streptococcus mutans wildtype, mupricin treated

treatment_protocol: Treated with 500 ng/ml of mupricin for 20 minutes at 37°C once OD600 of 0.3 was reached


title: Control SK+ 2

value: 4 days RNAi


title: SmutansUA159_Mupricin_rep3


title: Mip120 knock-down 1


title: SmutansUA159_Mupricin_rep2


title: SmutansUA159_Control_rep4

description: Streptococcus mutans wildtype, untreated control

source_name: S. mutans UA159 control condition

treatment_protocol: Untreated (control)


title: SmutansUA159_Control_rep3


title: Control SK+ 3


title: SmutansUA159_Control_rep2


title: SmutansJLrelA_Mupricin_rep3

description: Streptococcus mutans relA mutant, with mupricin treatment

source_name: S. mutans JLrelA mup treated

tag: Strain JLrelA

value: relA kockout

tag: strain UA159


title: Myb knock-down 1


title: Control SK+ 1

growth_protocol: Drosophila Kc cells were grown in suspension at 25°C in Schneider’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and  Antibiotic-Antimycotic solution (Invitrogen).

treatment_protocol:
Control :  RNAi in Drosophila Kc cells with a nonspecific RNA derived from a pBSK+ (named SK+).
Treatment : RNAi to deplete Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and  RBF2) and L(3)MBT.


title: SmutansJLrelA_Mupricin_rep1

description: Streptococcus mutans relA mutant, no mupricin treatment


title: SmutansJLrelA_Mupricin_rep2


title: SmutansJLrelA_Control_rep4

source_name: S. mutans JLrelA control condition


title: SmutansJLrelA_Mupricin_rep4