title: wild type at exponential phase vs wild type at stationary phase, rep3

city: East Lansing

last: Whittam

state: MI

address: 165 Food Safety & Tox

department: Microbiology

institute: Michigan State University

description: There are probes on the array that target three different E. coli genomes. Those that were specific for genomes other than Sakai were excluded from the analysis.

hyb_protocol: Arrays were cross-linked by exposure to 600 mJ UV before blocking in 1% SDS, 5× SSC, and 1 mg/mL BSA at 42°C for 1 hour. After blocking, arrays were washed 2× 5 min in 0.1× SSC and 2× 30 s in H2O. Dried arrays were placed into hybridization cassettes (TeleChem International, Sunnyvale, Calif.) and the cDNA samples were suspended in 10 mM EDTA, denatured at 95°C for 5 min and then mixed with 40 uL of SlideHyb buffer 1 (Ambion) and loaded under a coverslip onto the array. Hybridizations were carried out at 47°C for 16–18 h. After hybridization, arrays were washed in 2× SSC, 0.5% SDS 37C for 5 min, followed by 2× 5 min in 0.1× SSC, 0.1% SDS 37°C, and then 2 × 2.5 min in room temperature 0.1× SSC. Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).

data_processing: The microarray data were analyzed using R (v. 2.2.1) and the MAANOVA (v. 0.98.8) package.  Raw intensity values from replicate probes were averaged and log2 transformed after normalization with the pin-tip LOWESS method.  The normalized intensity values were fitted to a mixed model ANOVA considering array and biological replicates as random factors and dye, strain and growth phase as fixed factors.  The linear model tested was Y (intensity) = array + dye + strain (wild type or mutant) + growth phase (exponential or stationary) + strain*growth phase + sample (biological replicate) + error.  Significant differences in expression due to strain, growth phase and strain*growth phase were determined using the Fs test in MAANOVA which uses a shrinkage estimator for gene-specific variance components that makes no assumption about the variances across genes with 500 random permutations to estimate the p-values.  The q-value package in R was used for determining the false discovery rate (FDR).

organism: Escherichia coli

source_name: wild type cells grown to stationary phase of growth

label_protocol: Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM amino-allyl dUTP. 30uL C. cDNA was purified using PCRreactions were incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto stationary phase (OD600=1.0, 5.5h after inoculation)

extract_protocol: 5mL of culture was mixed with 5mL of hot acid phenol:chloroform. Samples were held at 65ºC with periodic shaking for at least 10 minutes before centrifuging at 4000 rpm for 20 min. Supernatant was extracted again with acid-phenol:chloroform and then with chloroform:isoamyl alcohol (24.1). RNA was precipitated overnight at –80ºC in 2.5V 100% ethanol and 1/10V 3M sodium acetate pH 5.2. RNA samples were purified and treated with DNase using the Rneasy kit (Qiagen).

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase - culture replicate 3

source_name: wild type cells grown to exponential phase of growth

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto exponential phase (OD600=0.25,~2.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential- culture replicate 3


title: wild type at exponential phase vs wild type at stationary phase, rep4

status: Public on Oct 11 2008

type: RNA

first: Thomas

middle: S

country: USA

scan_protocol: Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).

molecule: total RNA

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate 4

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 6


title: wild type at exponential phase vs wild type at stationary phase, rep5

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto sexponential phase (OD600=0.25, ~2.5h after inoculation)

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 5

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto stationary phase (OD600=1.0,5.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate 5


title: Mouse PUER exon 0h-1

city: Regensburg

last: Langmann

address: Franz-Josef-Strauss-Allee 11

department: University of Regensburg

country: Germany

institute: Institute of Human Genetics

description: Gene expression data from PU-ER cells without expression of PU.1.

hyb_protocol: The labeling assay yielded labeled single-stranded DNA that was fragmented and hybridized to Affymetrix Mouse Exon 1.0 ST Arrays (exon arrays) according to manufacturer’s protocols at the KFB Regensburg

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 Blank7G (Affymetrix).

data_processing:
The data were analyzed with Affymetrix Expression console and Genomatix ChipInspector using Affymetrix default analysis settings and global scaling as normalization method.
MoEx-1_0-st-v1.r2.dt1.mm8.core.mps

label: Biotin

organism: Mus musculus

source_name: PU-ER cells without expression of PU.1

label_protocol: Biotinylated sense-strand DNA for hybridization to Affymetrix exon arrays was prepared with the Affymetrix GeneChip whole transcript sense target labeling assay, using the standard 1,5 μg total RNA method according to the manufacturer’s protocols (Affymetrix, GeneChip WT terminal labelling kit 900671). Ribosomal RNA reduction was performed using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen, K1550-01).

growth_protocol: PUER cells were cultivated in IMDM supplemented with penicillin/streptomycin (10,000 U/ml), glutamin (200 mM),  ß-ME (50  M), mouse IL-3 (5 ng/ml, Biosource, Camarillo, CA, USA), puromycin (1  g/ml), and 10% FCS.

extract_protocol: Total RNA from the PUER cells was extracted according to the manufacter's instructions using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA)

treatment_protocol: To activate the PU.1-ER fusion protein, PUER cells were centrifuged and resuspended in medium containing 100nM tamoxifen [4-hydroxytamoxifen (OHT)]

tag: PU.1 -/- deficient myeloid progenitors with PU-ER fusion protein


title: wild type at exponential phase vs wild type at stationary phase, rep2

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate2

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 2


title: N11A Normal-frontal

city: Philadelphia

first: Alice

last: Chen-Plotkin

state: PA

address: 3600 Spruce Street 3rd Floor Maloney

department: Pathology and Neurology

institute: University of Pennsylvania

hyb_protocol: cRNA was hybridized for 16 hr at 45C on the Affymetrix Human U133A array.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip 3000 7G scanner.

data_processing: RMA

label: biotin

organism: Homo sapiens

source_name: N11A Normal-frontal

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 3 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

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

tag: postmortem brain


title: Male E9.5-3c

type: other

city: Geelong

first: Stephanie

middle: C

last: Bannister

state: Victoria

address: 5 Portarlington Road

department: Livestock Industries

country: Australia

institute: CSIRO

description:
small RNA <200nt
27spots excluded from data extraction: probe no see additional file sample21-ex.txt
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

hyb_protocol: CombiMatrix CustomArray 4x2k miRNA microarrays (design #4166) stored at room temperature prior to use.  Rehydrated and prehybridised according to CombiMatrix hybridisation and imaging protocol.  Hybridisation solutions prepared from concentrated, labelled miRNA samples adjusting volume of nuclease-free water (NF-H2O) used according to volume of miRNA sample.  Hybridisations carried out at 37degrees celcius in rotating hybridisation oven for 4hours or overnight.  Post-hybridisation wash steps and array preparation for imaging performed according to manufacturer's protocol.  Arrays were stripped following hybridisation and scanning according to manufacturer's protocol and stored in glad-wrap at 4degrees prior to re-hybridisation.  Individual array slides were stripped and re-hybridised between 4-10 times

scan_protocol: Arrays scanned using arrayWorx e microarray scanner (Applied Precision) and arrayWorx e scanning software.  Single Cy-5 channel used.  Resolution setting: High SNR 3.252um/pixel.  Focus adjusted manually per array.  Each array hybridisation was scanned at 5 exposure levels: a = 0.1sec; b = 0.2sec; c - 0.5sec; d = 1.0sec; e = 2.0sec.  Each exposure image saved as 24bit RGB colour image in TIFF format.

data_processing: CombiMatrix microarray imager software used to analyse scanned array images and extract data.  Briefly, chip design file #4166 opened as probe 1 and TIFF image opened at 100% size. Array template overlaid manually and data extracted for each individual array panel (Arrays 1-4).  Data for each individual array exported to text file and saved with array number, date, chip used and hybridisation number.  Data for arrays 1-4 imported into Microsoft Excel Spreadsheet template using an in-house macro, and background, subtraction, negative correction and normalisation was carried out using macro encompassing the following steps:  (1) Probe signal intensity values ranked from highest to lowest to identify 80th percentile value (2) 80th percentile value per sample subtracted from intensity value for each probe to give background-subtracted data set. (3) Negative numbers in background subtracted set made equal to zero (0). (4) Background subtracted values multipled by constant of 2 million (2.0E+6) to give normalised signal intensity.  Fold change analysis performed in Excel eg: by dividing sample 1 by sample 2 to give ratio sample1:sample2.

molecule: other

organism: Gallus gallus

source_name: Embryonic gonad

label_protocol: 500ng aliquot of miRNA sample was used per microarray hybridisation.  500ng volume of RNA sample was concentrated by vacuum drying using SpeedVac and resuspended in 17ul of Rnase-free water (Promega).  miRNA samples were labelled with ULS™-Cy5 using ULS™ Small RNA labelling kit (Kreatech Diagnostics).  This labels guanine (G) residues within miRNA sequence.  Therefore some G-bias in labelling. Protocol carried out as per manufacturers instructions.  Labelled samples purified using KreaPure columns (Kreatech Diagnostics) according to manufacturer's instructions and concentrated to less than 5.1ul using Microcon centrifugal filter columns (YM-30) (Microcon Bioseparations, Millipore) according to manufacturer's instructions.

extract_protocol: miRNA was extracted from pooled gonad lysates using the mirVana miRNA isolation kit (Ambion) protocol for enrichment of small RNAs <200nt, according to the manufacturer's instructions.  High-molecular weight (HMW) RNA fractions were also collected separately for all samples.  miRNA and HMW eluted in 100ul of RNase-free water (Ambion) pre-heated to 95degrees, quantified by nanodrop and stored at -20degrees celcius prior to microarray analysis.

tag: Sex

value: Male

tag: Stage

tag: Sexed

value: Manual

tag: exposure

tag: array chip #


title: Male E9.5-2e

description:
small RNA <200nt
6 spots excluded from data extraction: probe no; 271;305;590;711;849;903
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Female E6.5-2d

description:
small RNA <200nt
13 spots excluded from data extraction: probe no; 183;350;453;598;679;749;1016;1299;1300;1354;1355;1436;1437
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

value: Female

tag: sexed

value: PCR


title: Male E9.5-1-1d

description:
small RNA <200nt
1 spot excluded from data extraction: probe no; 543
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: N10A Normal-frontal

source_name: N10A Normal-frontal


title: Female E9.5-1-2e

description:
small RNA <200nt
30 spots excluded from data extraction: probe no see attached file sample23-ex.txt
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Female E6.5-1-2a

description:
small RNA <200nt
0 spots excluded from data extraction
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Female E9.5-1-1c

description:
small RNA <200nt
27spots excluded from data extraction: probe no see additional file sample22-ex.txt
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: N6A Normal-frontal

source_name: N6A Normal-frontal


title: wild type at exponential phase vs wild type at stationary phase, rep1

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto exponential phase (OD600=0.25, ~2.5h after inoculation)

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 1

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate 1


title: N5B Normal-cerebelum

source_name: N5B Normal-cerebelum


title: gadE mutant at stationary phase vs wild type at stationary phase, rep3

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto stationary phase (OD600=1.0, 5.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate 3

source_name: gadE mutant cells grown to stationary phase of growth

growth_protocol: gadE mutant cells were grown in MOPS minimal media upto stationary phase (OD600=1.0,5.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 3


title: gadE mutant at stationary phase vs wild type at stationary phase, rep4

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate 4

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 4


title: Male E9.5-1-2e

description:
small RNA <200nt
10 spots excluded from data extraction; 223;268;443;555;973;1056;1128;1176;1187;1323
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: gadE mutant at stationary phase vs wild type at stationary phase, rep1

growth_protocol: gadE muatant cells were grown in MOPS minimal media upto stationary phase (OD600=1.0,5.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 1


title: gadE mutant at exponential phase vs wild type at exponential phase, rep5

source_name: gadE mutant cells grown to exponential phase of growth

growth_protocol: gadE mutant cells were grown in MOPS minimal media upto exponential phase (OD600=0.25, ~2.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase- culture replicate 5

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto exponential phase (OD600=.25,~2.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 5


title: gadE mutant at stationary phase vs wild type at stationary phase, rep2

growth_protocol: gadE mutant cells were grown in MOPS minimal media upto stationary phase (OD600=1.0, 5.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 2

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase- culture replicate 2


title: N8C Normal-hippocampus

source_name: N8C Normal-hippocampus

tag: postmortem brain


title: gadE mutant at stationary phase vs wild type at stationary phase, rep5

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 5

tag: E. coli O157:H7 RIMD0509952 (Sakai) at stationary phase - culture replicate 5


title: N7B Normal-cerebellum

source_name: N7B Normal-cerebellum


title: Ts65Dn 19-20dpp (sample #3)

city: Bar Harbor

first: Laura

middle: G

last: Reinholdt

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

description: Male germ cells from Ts65Dn 19-20dpp (sample #3)

hyb_protocol: Illumina BeadChip hybridization protocol for 16 hours at 55 C.

scan_protocol: Illumina BeadChip scanning protocol using BeadStation 500 scanner.

data_processing: Images were checked for grid alignment and quantified using the BeadStudio software.

label_protocol: Illumina BeadChip labeling protocol.

extract_protocol: Qiagen RNeasy


title: N5A Normal-frontal

source_name: N5A Normal-frontal


title: Ts65Dn 19-20dpp (sample #2)

description: Male germ cells from Ts65Dn 19-20dpp (sample #2)


title: Female E6.5-1-1b

description:
small RNA <200nt
13 spots excluded from data extraction: probe no; 382;508;517;821;833;859;891;914;924;977;1155;1215;1316
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

tag: Sex

tag: Stage

tag: exposure


title: Male E6.5-6d

description:
small RNA <200nt
17 spots excluded from data extraction: probe no; 129;278;284;329;417;483;496;662;667;837;889;1003;1059;1122;1182;1196;1354;
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

tag: sexed

tag: array chip #


title: gadE mutant at exponential phase vs wild type at exponential phase, rep4

growth_protocol: O157:H7 wild type cells were grown in MOPS minimal media upto exponential phase (OD600=0.25, ~2.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase - culture replicate 4

growth_protocol: gadE mutant cells were grown in MOPS minimal media upto exponential phase (OD600=.25,~2.5h after inoculation).

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase- culture replicate 4


title: N10B Normal-cerebellum

source_name: N10B Normal-cerebellum


title: Male E6.5-3d

description:
small RNA <200nt
99 spots excluded from data extraction: probe no see additional file sample13-ex.txt
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Ts65Dn 16-17dpp (sample #3)

description: Male germ cells from Ts65Dn 16-17dpp (sample #3)


title: Male E6.5-2c

description:
small RNA <200nt
12 spots excluded from data extraction: probe no; 100;131;154;205;490;547;699;808;1112;1264;1280;1381
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: gadE mutant at exponential phase vs wild type at exponential phase, rep3

description: There are probes on the array that target three different E. coli genomes. Those that were specific for genomes other than Sakai were excluded from the analysis.

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase- culture replicate 3

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 3


title: N7A Normal-frontal

source_name: N7A Normal-frontal


title: N1C Normal-hippocampus

source_name: N1C Normal-hippocampus


title: N1B Normal-cerebellum

source_name: N1B Normal-cerebellum


title: N2A Normal-frontal

source_name: N2A Normal-frontal


title: gadE mutant at exponential phase vs gadE mutant at stationary phase, rep5

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 4


title: Ts65Dn 19-20dpp (sample #1)

description: Male germ cells from Ts65Dn 19-20dpp (sample #1)


title: Ts65Dn 16-17dpp (sample #2)

description: Male germ cells from Ts65Dn 16-17dpp (sample #2)


title: Male E6.5-1-1b


title: Ts65Dn 16-17dpp (sample #1)

description: Male germ cells from Ts65Dn 16-17dpp (sample #1)


title: Ts1Cje 19-20dpp (sample #2)

description: Male germ cells from Ts1Cje 19-20dpp (sample #2)


title: gadE mutant at exponential phase vs wild type at exponential phase, rep2

source_name: wild type cells grown to exponential phase of growth

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase - culture replicate 2

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase- culture replicate 2


title: gadE mutant at exponential phase vs gadE mutant at stationary phase, rep4


title: Ts1Cje 19-20dpp (sample #3)

description: Male germ cells from Ts1Cje 19-20dpp (sample #3)


title: Female E5.5-4e

description:
small RNA <200nt
25 spots excluded from data extraction:probe no see additional file Sample9-ex.txt
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Female E5.5-5e

description:
small RNA <200nt
95 spots excluded from data extraction: probe no see additional file - Sample-8ex.txt
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Female E5.5-1-2e

description:
small RNA <200nt
12 spots excluded from data extraction: probe no; 56; 118; 126; 158; 480; 605; 624; 658; 855; 1021; 1105; 1201
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Female E5.5-3d

description:
small RNA <200nt
14 spots excluded from data extraction: probe no; 236; 405; 435; 455; 494; 620; 954; 1023; 1132; 1320 1354; 1372; 1406; 1415
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: gadE mutant at exponential phase vs wild type at exponential phase, rep1

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase - culture replicate 1

tag: E. coli O157:H7 RIMD0509952 (Sakai) at exponential phase- culture replicate 1


title: gadE mutant at exponential phase vs gadE mutant at stationary phase, rep2

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 2.

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase- culture replicate 2.


title: Male E5.5-6e

description:
small RNA <200nt
15 spots excluded from data extraction: probe no; 258; 676; 697; 711; 755; 907; 916; 946; 957; 964; 1206; 1215; 1299; 1318; 1344;
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: gadE mutant at exponential phase vs gadE mutant at stationary phase, rep3

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 3


title: N1A Normal-frontal

source_name: N1A Normal-frontal

tag: postmortem brain


title: Ts1Cje 16-17dpp (sample #2)

description: Male germ cells from Ts1Cje 16-17dpp (sample #2)


title: gadE mutant at exponential phase vs gadE mutant at stationary phase, rep1

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at stationary phase- culture replicate 1

tag: E. coli O157:H7 RIMD0509952 (Sakai) gadE mutant at exponential phase- culture replicate 1


title: Male E5.5-2c

description:
small RNA <200nt
4 spots excluded from data extraction: probe no; 499, 717, 1036, 1075
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

tag: Sex

tag: Stage

tag: sexed

tag: exposure


title: WT diploid wild-type littermate control 19-20dpp (sample #3)

description: Male germ cells from diploid wild-type littermate control, WT, at 19-20dpp (sample #3)


title: Male E5.5-3e

description:
small RNA <200nt
16 spots excluded from data extraction: probe no; 83; 97; 127; 211; 213; 236; 293; 450; 491; 711; 911; 1073; 1136; 1179; 1245; 1270
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

tag: array chip #


title: Ts1Cje 16-17dpp (sample #3)

description: Male germ cells from Ts1Cje 16-17dpp (sample #3)


title: Male E5.5-1-2b

description:
small RNA <200nt
0 spots excluded from data extraction
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).


title: Ts1Cje 19-20dpp (sample #1)

description: Male germ cells from Ts1Cje 19-20dpp (sample #1)


title: Ts1Cje 16-17dpp (sample #1)

description: Male germ cells from Ts1Cje 16-17dpp (sample #1)


title: Exp-rpoS-Rep3

status: Public on Oct 10 2008

city: Hamilton

first: Herb

middle: E

last: Schellhorn

state: ON

address: 1280 Main St. W.,

department: Biology

country: Canada

institute: McMaster University

description: This study is to identify the RpoS regulon in MG1655 grown in M63 glucose (0.2%) minimal media in exponential growth (OD600=0.3) and stationary phase (OD600=1.5). RNA samples from wild type or rpoS mutants were extracted using acidic hot phenol method and hybridized to Affymetrix E. coli Antisense Genome Array.

hyb_protocol: Affymetrix protocol

scan_protocol: Affymetrix protocol

data_processing: GCRMA normalized

organism: Escherichia coli K-12

source_name: E. coli K12 grown in M63 glucose (0.2%) minimal media, sampled in exponential phase OD600=0.3

label_protocol: Affymetrix protocol

growth_protocol: Cells were grown in M63 glucose (0.2%) minimal media at 37oC at 200 rpm. Overnight cultures were inoculated with single, independent colonies in triplicate, incubated aerobically overnight at 37oC, and sub-cultured to a starting OD600 of 0.0001. Cultures were harvested at an OD600 = 0.3 after being maintained in exponential phase for at least eight generations.

extract_protocol: Acidic Hot Phenol

tag: MG1655 rpoS mutants


title: Exp-WT-Rep2

tag: MG1655 wild type


title: Male E5.5-1-1c

description:
small RNA <200nt
5 spots excluded from data extraction: probe numbers 61, 196, 1049, 1200, 1239.
Gonads were dissected from chicken embryos at embryonic days 5.5, 6.5 and 9.5 in PBS treated with diethylpyrocarbonate (DEPC).  Gonad pairs from individual E5.5 and E6.5 embryos were lysed in 30ul of miRNA lysis/binding buffer (Ambion mirVana miRNA isolation kit), vortexed and incubated at 4 degrees celcius until sexing was carried out. Then, gonad lysates were pooled by sex for miRNA extraction.  E9.5 embryos were sexed manually during dissection given asymmetric development of female gonads can be used to visually distinguish male and female embryos.  E9.5 gonads were pooled directly by sex during dissection, into 300ul of miRNA lysis/binding buffer on ice/4 degrees celcius until miRNA extraction.
PCR sexing to distinguish male and female embryos was carried out as previously described (Clinton et. al., 2001, Sexing chick embryos: a rapid and simple protocol, Br. Poult. Sci. v42(1), pp 134-138).  Small piece of hind-limb tissue was taken from each E5.5 and E6.5 embyo and digested in PCR digestion buffer (50mM KCl; 10mM Tris HCl (pH 8.3); 0.1mg/ml gelatin; 0.45% NP40; 0.45% Tween-20; 200ug/ml Proteinase K) at 55 degrees celcius for minimum of 30 minutes, then 95 degrees celcius for 10 minutes.  Digested tissue samples cooled to room temperature.  1ul of digested sample added to each PCR (see paper ref. for reaction mix).  RedTaq Polymerase (Sgima) used to amplify products - 1ul/reaction.  PCR products routinely run on 1.5-1.8% TBE agarose gel containing GelRed (Biotium).  Female positive samples give 2 bands; W-specific band (larger) and 18S control band (smaller).  Male positive samples give only 18S control band (no W chromosome in males).

source_name: Embryonic gonad


title: WT diploid wild-type littermate control 19-20dpp (sample #1)

description: Male germ cells from diploid wild-type littermate control, WT, at 19-20dpp (sample #1)

source_name: Male germ cells from WT diploid wild-type littermate control at 19-20dpp (sample #1)


title: Exp-WT-Rep1


title: WT diploid wild-type littermate control 19-20dpp (sample #2)

description: Male germ cells from diploid wild-type littermate control, WT, at 19-20dpp (sample #2)


title: WT diploid wild-type littermate control 16-17dpp (sample #2)

description: Male germ cells from diploid wild-type littermate control, WT, at 16-17dpp (sample #2)

source_name: Male germ cells from WT diploid wild-type littermate control at 16-17dpp (sample #2)


title: Stat-WT-Rep1

source_name: E. coli K12 grown in M63 glucose (0.2%) minimal media, sampled in stationary phase OD600=1.5

growth_protocol: Cells were grown in M63 glucose (0.2%) minimal media at 37oC at 200 rpm. Overnight cultures were inoculated with single, independent colonies in triplicate, incubated aerobically overnight at 37oC, and sub-cultured to a starting OD600 of 0.0001. Cultures were harvested at an OD600 = 1.5.


title: Stat-rpoS-Rep1


title: WT diploid wild-type littermate control 16-17dpp (sample #3)

description: Male germ cells from diploid wild-type littermate control, WT, at 16-17dpp (sample #3)

source_name: Male germ cells from WT diploid wild-type littermate control at 16-17dpp (sample #3)


title: Exp-rpoS-Rep2


title: Stat-WT-Rep2


title: Stat-rpoS-Rep3


title: Exp-rpoS-Rep1


title: WT diploid wild-type littermate control 16-17dpp (sample #1)

description: Male germ cells from diploid wild-type littermate control, WT, at 16-17dpp (sample #1)

source_name: Male germ cells from WT diploid wild-type littermate control at 16-17dpp (sample #1)


title: Exp-WT-Rep3


title: Stat-rpoS-Rep2


title: Stat-WT-Rep3


title: OP9_MG-U74C

city: Machida-shi

first: Kenji

last: Nagao

state: Tokyo

address: 3-6-6, Asahi-machi,

department: Innovative Drug Research Laboratories

country: Japan

institute: Kyowa Hakko Kirin Co., Ltd.

description: no additional information

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on the Mouse Genome MG-U74A, B, C Probe GeneChip array using Affymetrix Hybridization Oven 640. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using Affymetrix GeneChip Scanner 3000.

data_processing: GeneChip software was used to determine the average difference (AD) in the levels of gene expression among genes on the array. The mean AD for 3’-terminal probe sets corresponding to four constitutively expressed genes (beta-actin, GAPDH, pyruvate carboxylase and transferrin receptor) were calculated, and hereafter we use STD to refer to the mean AD of the control probe set. To normalize staining intensity among chips, the AD values for all genes on a given chip were divided by the ratio of the STD for each chip to the average STD for all chips. Normalized AD values less than 0.1 were set to 0.1. Then, the dataset was sorted by the ratio of the mean AD of each gene in a target group to that in a reference group in order to identify highly expressed genes from within an experimental group.

source_name: hematopoiesis supportive stromal cells derived from calvaria of newborn mice

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA.

extract_protocol: RNeasy mini-kit (Qiagen, Chatsworth, CA) extraction of total RNA was performed according to the manufacturer's instructions.

tag: Strain:(C57BL/6 x C3H)F2 -op/op


title: OP9_MG-U74A

description: no additional information


title: AGM-s3-A9_MG-U74A

description: no additional information

source_name: hematopoiesis supportive stromal cells derived from AGM region


title: AGM-s3-A7_MG-U74A

description: no additional information

source_name: hematopoiesis non-supportive stromal cells derived from AGM region


title: OP9_MG-U74B

description: no additional information


title: AGM-s3-A7_MG-U74B

description: no additional information


title: AGM-s3-A9_MG-U74B

description: no additional information


title: AGM-s3-A7_MG-U74C

description: no additional information


title: AGM-s3-A9_MG-U74C

description: no additional information


title: NHP8/DNp53 (Senescent) vs Young NHP8 Cells_rep3

status: Public on Oct 09 2008

city: Smithville

first: Dean

last: Tang

state: TX

address: 1808 Park road 1C

department: Carcinogenesis

institute: U.T M. D. Anderson Cancer Center

description: The current array analysis was carried to study the changes in the gene expression profile of NHP8/DNp53 cells compared to yougn NHP cells.

hyb_protocol: Manufacturer's protocol available at http://www.chem.agilent.com/temp/rad71A57/00001389.PDF

scan_protocol: Scanning and data acquisition were performed using Agilent scanner and feature extraction protocol GE2-v5_91 (available at www.agilent.com).

data_processing:
The data was acquired using Agilent's feature extraction protocol
GE2-v5_91 with inbuilt Lowess normalization feature and Logratio
algorithms, for details visit www.agilent.com.

source_name: Young NHP8 cells

label_protocol:
Labelling was done according to manufacturer's protocol available at
http://www.chem.agilent.com/temp/rad71A57/00001389.PDF

growth_protocol: NHP cells were mostly cultured on collagen-coated dishes in serum/androgen-free, partially defined PrEBM medium containing insulin, EGF, bovine pituitary extract (BPE; Invitrogen, Carlsbad, CA), and antibiotics.

extract_protocol:
Total RNA was extracted from NHP cells (see Fig. 7A) using an RNeasy
RNA-purification kit (Qiagen, Valencia, CA), including on-column DNase
digestion to completely remove contaminating genomic DNA. RNA
concentrations were determined in NanoDrop and the quality
assessed using a BioAanalyzer.

tag: young NHP8 cells at passage-3 (control)

source_name: NHP8/DNp53 (senescent cells)

tag: NHP8 cells infected with retrovirus encoding DN(dominant negative)p53 and at passage-6


title: NHP8/p16sh + hTERT (immortalized) vs young NHP8 cells_rep3

description: The current array analysis was carried to study the changes in the gene expression profile of NHP8/p16sh + hTERT (immortalized) cells compared to young NHP8 cells.

data_processing:
The data was acquired using Agilent's feature extraction protocol
GE2-v5_91 with inbuilt Lowess normalization feature and Logratio
algorithms, for details visit www.agilent.com

source_name: NHP8/p16sh + hTERT (immortalized) cells

tag: NHP8 cells infected with retrovirus encoding p16 small hairpin RNA and hTERT and at passage-21


title: S0983_Hind240

type: genomic

city: Newcastle upon Tyne

first: Michael

middle: Stewart

last: Jackson

address: Central Parkway

department: Institute of Genetic Medicine

country: United Kingdom

institute: Newcastle University

hyb_protocol: All samples were hybridised using the Affymetrix Mapping 100K v1_450 protocol according to manufacturers recommendations (Affymetrix UK Ltd., High Wycome, UK).

scan_protocol: All samples were scanned using an Affymetrix GeneChip Scanner 3000

data_processing: Data was processed with GDAS 3.0 software using default settings.

molecule: genomic DNA

source_name: peripheral blood

label_protocol: The amplified products were fragmented and end labelled with a fluorescent tag according to manufacturers recommendations (Affymetrix UK Ltd., High Wycome, UK).

extract_protocol: DNA was extracted using a phenol/chloroform/isoamyl alcohol extraction after proteinase K treatment. DNAs were digested with HindIII and ligated to appropriate adapters prior to PCR amplification. Where DNA quantity was limited, a whole genome amplification step (Multiple Displacement Amplification) was performed prior to digestion according to manufacturer’s instructions (Quiagen Inc., Valencia, CA).

treatment_protocol: All samples were snap frozen and stored at -80oC prior to extraction

tag: Gender

value: male

tag: Age

value: 4 years


title: NHP8/p16sh + hTERT + DNp53 (immortalized) vs young NHP8 cells_rep2

description: The current array analysis was carried to study the changes in the gene expression profile of NHP8/p16sh + hTERT + DNp53 (immortalized) cells compared to young NHP8 cells.

tag: NHP8 cells infected with retrovirus encoding p16 small hairpin RNA, hTERT and DN(dominant negative)p53 and at passage-21


title: S0984_Hind240

value: 2 years


title: NHP8/DNp53 (Senescent) vs Young NHP8 Cells_rep1

data_processing:
The data was acquired using Agilent's feature extraction protocol
GE2-v5_91 with an inbuilt Lowess normalization feature and Logratio
algorithms, for details visit www.agilent.com.


title: 23A4_v_W83

city: Columbus

first: CHRISTINA

last: IGBOIN

state: OH

address: 305 W 12th Ave

institute: Ohio State University

description: Sample row represents 2 flip dye hybridizations with 1 repeat (3 raw data files)

hyb_protocol: Hybridization of labeled DNA to the microarray slides was performed according to TIGR supplied protocols. The slides were prehybridized at 42°C in 5X SSC, 0.1% SDS, 1% BSA for 2 hours and then washed with shaking using 2L of water. After a further 2 min wash in isopropyl alcohol, the slides were dried by centrifugation at 1,100RPM for at least 5 mins. The labeled mixed DNA (test and control) was resuspended in 60ml of hybridization buffer (50% formamide, 5X SSC, 0.1% SDS, 0.6mg/ml salmon sperm DNA), denatured at 95°C for 10 mins., and applied to the slides. Hybridization was carried out under LifterSlips (Erie Scientific), at 42°C in a sealed hybridization chamber for 20 hours. Following hybridization, the slides were washed 3 times in low stringency wash (2X SSC, 0.1% SDS, 55°C, 5 min), 2 times in medium stringency wash (0.1X SSC, 0.1% SDS, 5 min), 2 times in high stringency wash (0.1X SSC, 5 min) and once with water (1 min). Flip-dye replicates were performed for each test strain.

scan_protocol: The microarrays were scanned using a 480 scanner (Affymetrix) or a GenePix 4000 scanner (Axon Instruments), and slide images were processed with GenePix Pro 6.0 (Axon Instruments).

data_processing: Spots that could not be identified by the software, and by visual inspection, as well as spots with slide abnormalities or hybridization aberrations (dust, scratches etc.), were excluded from further analysis. For further analysis, the GenePix output data were uploaded into Gene Traffic 3.2. (Iobion Informatics). Spots were filtered for low signal (control signal < background or control signal <100 units), and the data were normalized using LOWESS intensity based normalization. The log2  (test/control) ratios were computed, and averaged across replicates (within and then between slides). Only genes with at least 2 valid measurements in each of the flip dye hybridizations were included in the final data set.

organism: Porphyromonas gingivalis

source_name: strain W83, strain 23A4

label_protocol: Test and control DNA’s were labeled according to TIGR supplied protocols. Random labeling of 4mg of test and control genomic DNA was performed in 50ml reactions consisting of 20 units of Klenow enzyme (New England Biolabs), 0.2mg/ml random hexamers (Invitrogen), 1X Klenow buffer and 0.2mM dNTP/ aa-dUTP mix (2:1). The reactions were incubated for 16 hours at 37°C, then the enzyme was inactivated by adding 5ml of 0.5M EDTA (pH 8). The reactions were purified using Qiagen PCR purification columns, with phosphate wash (5mM KPO4 pH 8, 80% ethanol) and phosphate elution (4mM KPO4 pH 8.5) buffers in place of the Qiagen supplied buffers. The eluted DNA was dried in a speed vac, resuspended in 4.5ml of 0.1M sodium carbonate (pH 9.3), and coupled with Cy3 or Cy5 dyes (4.5ml in DMSO) for 5 hours. Coupling was stopped by the addition of 35ml of 100mM sodium acetate (pH 5.2), and the reactions were purified using Qiagen PCR purification columns. The DNA was eluted in 60ml of tris chloride (pH 8), and the labeling efficiency was assessed using a Nanodrop ND-1000 spectrophotometer (Nanodrop). Labeled test and control DNA’s were mixed and dried down in a speed vac.

growth_protocol: The bacteria were grown on Brucella blood agar (Anaerobe Systems), at 37°C in an anaerobic chamber (85% N2, 10% H2, 5% CO2).

extract_protocol: Genomic DNA was isolated using cetyltrimethly ammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol extractions, followed by ethanol precipitation. Plate grown bacteria were suspended in 800 ml of 1% sodium dodecyl sulfate in tris EDTA, and incubated at 95°C for 5 min with vortexing. 20 ml of 3% proteinase K was added, and the samples were incubated at 37°C overnight. The samples were treated with 20 ml RNase A (20 mg/ml) at RT for 5 min, then100 ml of NaCl (5M) and 80 ml of CTAB/NaCl solution were added. The samples were vortexed, and incubated at 65°C for 10 min, then 500 ml of phenol: chloroform: isoamylalcohol (25:24:1) was added. The samples were vortexed thoroughly, centrifuged at 13,500 RPM for 5 min and the upper aqueous phase was transferred to a new tube. The phenol: chloroform: isoamylalcohol extraction was repeated until no more cellular debris was recovered in the organic phase. 0.1 volumes of sodium acetate (3M, pH 5.2), and 2 volumes of ice- cold ethanol were added to the aqueous phase, and precipitated DNA was pelleted at 13,500 RPM for 10 min at 4°C. The pelleted DNA was washed with 200 ml ice-cold 70% ethanol, dried in a speed vac and then dissolved in 10mM tris chloride (pH 8).

tag: originally isolated from individual with oral infection

source_name: strain 23A4, strain W83


title: S0579_Hind240

value: female

value: 1 year


title: S0578_Hind240

value: 10 years


title: NHP8/p16sh (near senescent) vs young NHP8 cells_rep3

description: The current array analysis was carried to study the changes in the gene expression profile of NHP8/p16sh (near-senescent) cells compared to young NHP cells.

source_name: NHP8/p16sh (near senescent) cells

tag: NHP8 cells infected with retrovirus encoding p16 small hairpin RNA and at passage-9


title: A7A1_v_W83

description: Sample row represents 2 flip dye hybridizations (2 raw data files)

source_name: strain W83, strainA7A1

source_name: strain A7A1, strain W83


title: S0577_Hind240

value: 1.5 year


title: Male E6.5-1-2a

source_name: Embryonic gonad


title: S0529_Hind240

source_name: primary tumour

tag: MYCN amp.:no

tag: 1p del

value: no

tag: 11q del

tag: 17q gain

value: mixed


title: NHP8/p16sh + hTERT + DNp53 (immortalized) vs young NHP8 cells_rep3

source_name: NHP8/p16sh + hTERT + DNp53 (immortalized) cells


title: NHP8/DNp53 (Senescent) vs Young NHP8 Cells_rep2


title: NHP8/p16sh + hTERT (immortalized) vs young NHP8 cells_rep2


title: W50_v_W83

source_name: strain W83, strain W50

source_name: strain W50, strain W83


title: NHP8/hTERT (Senescent) vs Young NHP8 cells_rep3

description: The current array analysis was carried to study the changes in the gene expression profile of NHP8/hTERT (senescent) cells compared to young NHP cells.

scan_protocol: Scanning and data acquisition was performed using Agilent scanner and  feature extraction protocol GE2-v5_91 (available at www.agilent.com).

data_processing: The data was acquired using Agilent's feature extraction protocol GE2-v5_91 with inbuilt Lowess normalization feature and Logratio algorithms, for details visit www.agilent.com

label_protocol: Labelling was done according to manufacturer's protocol available at http://www.chem.agilent.com/temp/rad71A57/00001389.PDF

extract_protocol:
Total RNA was extracted from NHP cells (see Fig. 7A) using an RNeasy RNA-purification kit (Qiagen, Valencia, CA), including on-column DNase digestion to completely remove contaminating genomic DNA. RNA concentrations were determined in NanoDrop and the quality
assessed using a BioAanalyzer.

source_name: NHP8/hTERT (senescent) cells

tag: NHP8 cells infected with retrovirus encoding hTERT and at passage-6


title: NHP8/p16sh + hTERT (immortalized) vs young NHP8 cells_rep1


title: NHP8/hTERT (Senescent) vs Young NHP8 cells_rep2


title: NHP8/p16sh + hTERT + DNp53 (immortalized) vs young NHP8 cells_rep1


title: NHP8/p16sh (near senescent) vs young NHP8 cells_rep2


title: 49417_v_W83

source_name: strain W83, strain  49417

source_name: strain  49417, strain W83


title: 381_v_W83

description: Sample row represents 2 flip dye hybridizations with 2 repeats (4 raw data files)

source_name: strain W83, strain381

source_name: strain  381, strain W83


title: 33277_v_W83

source_name: strain W83, strain33277

source_name: strain  33277, strain W83


title: NHP8/p16sh+DNp53 (pre-senescent) vs young NHP8 cells_rep2

description: The current array analysis was carried to study the changes in the gene expression profile of NHP8/p16sh + DNp53 (pre-senescent) cells compared to young NHP8 cells.

source_name: Young NHP8 cells

source_name: NHP8/p16sh+DNp53 (pre-senescent) cells

tag: NHP8 cells infected with retrovirus encoding p16 small hairpin RNA and DN(dominant negative)p53 and at passage-9


title: S0576_Hind240

value: <1 year

tag: Stage

value: yes

tag: 17q gain yes


title: S0571_Hind240

tag: MYCN amp.:yes


title: S0575_Hind240

value: 2.5 years


title: S0573_Hind240


title: S0528_Hind240


title: NHP8/p16sh+DNp53 (pre-senescent) vs young NHP8 cells_rep3

tag: young NHP8 cells at passage-3 (control)


title: NHP8/hTERT (Senescent) vs Young NHP8 cells_rep1

data_processing: The data was acquired using Agilent's feature extraction protocol GE2-v5_91 with an inbuilt Lowess normalization feature and Logratio algorithms, for details visit www.agilent.com


title: S0527_Hind240

value: 8 years


title: S0574_Hind240


title: S0570_Hind240


title: S0572_Hind240


title: S0445_Hind240


title: S0982_Hind240


title: S0443_Hind240

tag: 11q del:no


title: S0444_Hind240


title: S0525_Hind240


title: S0524_Hind240


title: NHP8/p16sh (near senescent) vs young NHP8 cells_rep1

source_name: NHP8/p16sh (near senescent cells)


title: PBMC-Stenotic-Rep26 (17-0748)

status: Public on Oct 08 2008

city: CA

first: James

middle: A.

last: Wingrove

state: CA

address: 2500 Faber Place

institute: CardioDx, Inc.

hyb_protocol: Labeled RNAs were hybridized using the Agilent protocol (Agilent, version 4.0.1) with the following exception: labeled reference samples were pooled in 1.06X Hybridization Buffer and 2X Blocking Buffer, the pooled samples were heated to 65 C for 5 minutes and then centrifuged at 13,000 rpm for 10 minutes

scan_protocol: Data was scanned on an Agilent Scanner (G2505B) and extracted using Agilent Feature Extraction Software (version 8.5.1.1)

data_processing:
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
Data was imported into GeneSpring (Agilent, v 7.3.1) and normalized using the AgilentFE normalization scenario.  Retained features had a present or marginal call in at least one sample.

source_name: PBMC - Stenotic

label_protocol: 100 ng of each patient PBMC RNA was labeled with Cy5 using the Low RNA Input Fluorescent Linear Amplification Kit from Agilent (PN 5184-3523), following the recommended protocol.  In parallel, 100 ng (per hybridization) of commercially available reference PBMC RNA (Clontech, PN 636580) was Cy3 labeled using the same protocol.

growth_protocol: Whole blood (16ml) was collected prior to angiography (BD Vacutainer® CPT); mononuclear cells were processed immediately following collection.

extract_protocol: RNA was extracted using a modification of the RNeasy method (Qiagen)

treatment_protocol: PBMC preps were processed using RLT buffer (Qiagen) prior to freezing at -80C

tag: Stenotic

source_name: PBMC Pool

tag: PBMC Pool - Clontech (PN # 636580)


title: NHP8 near senescent/senescent vs young_rep3

description: The current array analysis was carried to study the changes in the gene expression profiles of near senescent NHP cells compared to young NHP cells.

source_name: Normal human prostate (NHP8) young cells

source_name: NHP8 near senescent/senescent cells

tag: NHP8 cells at passage-5


title: PBMC-Stenotic-Rep27 (17-0722)


title: NHP8/p16sh+DNp53 (pre-senescent) vs young NHP8 cells_rep1


title: PBMC-Stenotic-Rep25 (17-1057)


title: S0442_Hind240

source_name: primary tumour

tag: Gender

tag: Age


title: PBMC-Stenotic-Rep22 (17-1077)


title: PBMC-Stenotic-Rep11 (17-0917)


title: PBMC-Stenotic-Rep19 (17-0815)


title: PBMC-Stenotic-Rep14 (17-0758)


title: PBMC-Stenotic-Rep16 (17-0958)


title: PBMC-Stenotic-Rep24 (17-1086)


title: PBMC-Stenotic-Rep23 (17-0954)


title: PBMC-Stenotic-Rep7 (17-1037)


title: PBMC-Stenotic-Rep17 (17-0951)


title: PBMC-Stenotic-Rep20 (17-1089)


title: NHP8 near senescent/senescent vs young_rep2


title: PBMC-Stenotic-Rep15 (17-0963)


title: PBMC-Stenotic-Rep21 (17-1083)


title: S0523_Hind240

growth_protocol: Not Applicable


title: PBMC-Stenotic-Rep13 (17-0808)


title: PBMC-Stenotic-Rep9 (17-0924)


title: PBMC-Stenotic-Rep6 (17-1043)


title: PBMC-Stenotic-Rep1 (17-0736)


title: PBMC-Stenotic-Rep8 (17-0994)


title: PBMC-NonStenotic-Rep12 (17-0842)

source_name: PBMC - Non Stenotic

tag: Non Stenotic


title: PBMC-Stenotic-Rep5 (17-0866)


title: PBMC-NonStenotic-Rep11 (17-1068)


title: PBMC-Stenotic-Rep3 (17-0915)


title: PBMC-NonStenotic-Rep9 (17-0828)


title: PBMC-NonStenotic-Rep14 (17-0750)


title: PBMC-Stenotic-Rep4 (17-0913)


title: NHP8 near senescent/senescent vs young_rep1


title: PBMC-Stenotic-Rep18 (17-0822)


title: PBMC-Stenotic-Rep12 (17-0916)


title: PBMC-Stenotic-Rep2 (17-0727)


title: PBMC-NonStenotic-Rep10 (17-0813)


title: PBMC-NonStenotic-Rep13 (17-0967)


title: 13256501 - Control vs DGPP 18:1 300(u)M

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

country: France

institute: INRA

description: The specific plant phosphorylated form of phosphatidic acid (PA), diacyglycerol pyrophosphate (DGPP) was recently shown to be a second messenger in abscisic acid (ABA) signaling. The aim of the project is to identify among the set of ABA-regulated genes the ones also regulated by DGPP and/or PA.

hyb_protocol: DGPP 18:1 300(u)M Cy5 / Control Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 3.0, Cy3:pmt voltage 532nm,650V,laser power 100%, Cy5:635nm,pmt voltage 620V,laser power 100%

data_processing: The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.

organism: Arabidopsis thaliana

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: cell culture - Cells in suspension Media : Jouanneau and Peaud-Lenokl (Jouanneau and Peaud-Lenokl, 1967) Temperature : 22°C Light : 40 umol photon s-1 m-2

extract_protocol: Pool of extract, DGPP 18:1 set1:0.5ug, DGPP 18:1 set2:0.5ug, DGPP 18:1 set3:0.5ug.

treatment_protocol: Name:DGPP 18:1 - compound based treatment - compound addition,dgpp 18:1:quantity 300000mM time 3hour . Five ml of 3 days-old suspension cells was incubated with lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4degre Celsius, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted.

tag: Arabidopsis thaliana (columbia)  - age:3day

source_name: Control

growth_protocol: cell culture - Cells in suspension. Media : Jouanneau and Peaud-Lenokl (Jouanneau and Peaud-Lenokl, 1967) Temperature : 22°C Light : 40 umol photon s-1 m-2

extract_protocol: Pool of extract, Control set1:0.5ug, Control set2:0.5ug, Control set3:0.5ug.

treatment_protocol: no treatment


title: PBMC-NonStenotic-Rep3 (17-0920)


title: PBMC-NonStenotic-Rep2 (17-0889)


title: PBMC-NonStenotic-Rep6 (17-0948)


title: 13256507 - DGPP 8:0 300(u)M vs Control

hyb_protocol: Control Cy5 / DGPP 8:0 300(u)M Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: Pool of extract, DGPP 8:0 set1:0.5ug, DGPP 8:0 set2:0.5ug, DGPP 8:0 set3:0.5ug.

treatment_protocol: Name:DGPP 8:0 - compound based treatment - compound addition,dgpp 8:0:quantity 300000mM time 3hour . Five ml of 3 days-old suspension cells was incubated with lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4degre Celsius, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted.


title: PBMC-NonStenotic-Rep7 (17-0933)


title: 13256502 - Control vs PA 8:0 300(u)M

hyb_protocol: PA 8:0 300(u)M Cy5 / Control Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: Pool of extract, PA 8:0 set1:0.5ug, PA 8:0 set2:0.5ug, PA 8:0 set3:0.5ug.

treatment_protocol: Name:PA 8:0 - compound based treatment - compound addition,pa 8:0:quantity 300000mM time 3hour . Five ml of 3 days-old suspension cells was incubated with lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4degre Celsius, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted.


title: PBMC-NonStenotic-Rep1 (17-0711)


title: 13256508 - DGPP 18:1 300(u)M vs Control

hyb_protocol: Control Cy5 / DGPP 18:1 300(u)M Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13256746 - ABA 10-5M vs Control

hyb_protocol: Control Cy5 / ABA 10-5M Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: ABA 10-5M

extract_protocol: Pool of extract, ABA 10-5M set1:0.5ug, ABA 10-5M set2:0.5ug, ABA 10-5M set3:0.5ug.

treatment_protocol: Name:ABA 10-5M - compound based treatment - compound addition,aba:quantity 0.01mM time 3hour . Five ml of 3 days-old suspension cells was incubated with ABA for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4degre Celsius, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted.


title: PBMC-NonStenotic-Rep8 (17-0929)


title: 13256510 - PA 18:1 300(u)M vs Control

hyb_protocol: Control Cy5 / PA 18:1 300(u)M Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

treatment_protocol: treatment

extract_protocol: Pool of extract, PA 18:1 set1:0.5ug, PA 18:1 set2:0.5ug, PA 18:1 set3:0.5ug.

treatment_protocol: Name:PA 18:1 - compound based treatment - compound addition,pa 18:1:quantity 300000mM time 3hour . Five ml of 3 days-old suspension cells was incubated with lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4degre Celsius, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted.


title: 13256500 - Control vs DGPP 8:0 300(u)M

hyb_protocol: DGPP 8:0 300(u)M Cy5 / Control Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13256505 - Control vs PA 18:1 300(u)M

hyb_protocol: PA 18:1 300(u)M Cy5 / Control Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: PBMC-NonStenotic-Rep4 (17-0810)


title: 13256509 - PA 8:0 300(u)M vs Control

hyb_protocol: Control Cy5 / PA 8:0 300(u)M Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: PBMC-NonStenotic-Rep5 (17-0985)