title: 14286377 - WT leaves Repet3 vs WT + MTX leaves Repet3

description: Folates and 1C metabolism

hyb_protocol: WT + MTX leaves Repet3 Cy5 / WT leaves Repet3 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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: WT + MTX leaves Repet3

extract_protocol: WT + MTX leaves Repet3:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

value: MTX

value: leaves

source_name: WT leaves Repet3

extract_protocol: WT leaves Repet3:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.


title: BrdU CTRL UBQ ChIP-chip replicate 2

status: Public on Aug 01 2013

type: genomic

city: Charlestown

first: Johnathan

last: Whestine

state: MA

address: RM7103 Bldg 149 13th Street

department: Cancer Center

country: USA

institute: MGH

description:
BrdU ChIP-chip in GFP and ubiquitin  overexpressing HEK293T cells
Also included is the Normalized data used to generate Figure 5.txt file.  This file contains the data used to generate the graph to in Figure 5 of Van Rechem et al. JBC 2011.  It is the smoothed, averaged BrdU signal intensities in the 2 megabases surrounding JMJD2A binding sites and randomly selected binding sites.

hyb_protocol: The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer using the Roche/Nimblegen Hybridization kit following the manufacturers instructions.  Hybridization was performed in Maui hybridization stations for 20h at 42C, and then washed in Roche/Nimblegen array wash buffers I, II, and IIIaccording to the manufacturer's protocols.(http://www.nimblegen.com/products/lit/lit.html)

scan_protocol: Arrays were scanned on Roche MS200 scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).

data_processing: Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.6 ChIP data extraction.  Raw BrdU ChIP intensity values in each channel were log-transformed (based 2) followed by quantile normalization. The normalization formula was determined based on intensity values at the 12,900 control probes printed on the Nimblegen array and applied to adjust values at all other probes. After normalization, the replicates were averaged for each experiment. We then averaged the normalized BrdU intensities around 2MB regions centered at the JMJD2A peak locations. Due to the incomplete coverage of the Nimblegen array, we excluded those regions that were less than 50% covered by the microarray, corresponding to 380 peaks. The averaged profile was further smoothed by moving average over sliding windows of 50 kb wide each at 1kb step size. For comparison, we randomly selected the same number of regions from the genome and obtained an average profile using the same procedure. For each channel, the mean intensity level was subtracted to further remove any potential artifact. We used a one sided paired t-test to determine whether the difference was statistically significant.

molecule: genomic DNA

organism: Homo sapiens

source_name: BrdU ChIP DNA from HEK293T cells overexpressing GFP and Ubiquitin

label_protocol: ChIP DNA  was amplified by WGA 2 Kit (Sigma) following the manufacturers protocol.  1 µg of amplified ChIP or Input DNA was directly labeled using Roche/Nimblegen Labeling dual color Labeling kits with random priming with Cy3 and Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).

growth_protocol: HEK293T cells were cultured for 48 hours in DMEM with 10% FBS.

extract_protocol: Subconfluent 10cm dishes (~3x10^6 cells) were washed off the plate in culture media. Cells were lysed in RIPA buffer for 20 minutes on ice and treated with RNAseA for 1 hr at 37 ˚C.  After RNase cells were treated with proteinase K for at least 4 hrs at 55˚C.   Genomic DNA was purified by phenol/chloroform extraction and ethanol precipitation.  DNA was sheared to an average size of 300 bp using a cup horn at 4 degrees C (Misonix).   DNA was denatured by heat at 95˚C for 10 minutes and immediately chilled on ice.  2 μg of DNA was incubated with 30 μl anti-BrdU (BD Biosciences #347580 lot: 57267) for 2hrs at 4˚C.  Immunocomplexes werecaptured with magnetic protein G beads.  Beads were washed 5x with PBS with 0.5% Triton-X100.  DNA was eluted in PBS with 1% SDS and 100ug/ml proteinase K.  DNA and equivalent input DNA were purified by Promega SV wizard DNA purification columns following the manufacturers protocol.  Confirmation of BrdU IP was made at Chr1 Sat2 by qPCR.

treatment_protocol: Cells were treated with 10μM BrdU for 1 hr prior to harves

tag: cell type

value: HEK293T GFP

tag: cell line

tag: chip antibody

tag: treatment


title: JMJD2A ChIP-chip replicate 1

description:
ChIP-chip JMJD2A in GFP-JMJD2A overexpressing HEK293T cells
Also included is the Normalized data used to generate Figure 5.txt file.  This file contains the data used to generate the graph to in Figure 5 of Van Rechem et al. JBC 2011.  It is the smoothed, averaged BrdU signal intensities in the 2 megabases surrounding JMJD2A binding sites and randomly selected binding sites.

data_processing: Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.6 ChIP data extraction.  The pair files were used (and indicated as appropriate replicates) with MA2C software for normalization and peak calling.  A total of 2,408 JMJD2A peak locations in the JMJD2A overexpressed cells were detected by using the MA2C software (p-value cutoff =1.0E-3).

source_name: JMJD2A ChIP DNA from HEK293T cells overexpressing GFP-JMJD2A

extract_protocol: Subconfluent 15cm dishes (~5x10^6 cells) were fixed with 1% formaldehyde in culture medium for 15 min at 37 degrees C followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed. The crosslinked chromatin was sheared to an average size of 300 bp using a cup horn at 4 degrees C (Misonix). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (20 µg) was RNAse A treated for 1 hr and diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and was then incubated overnight with anti-JMJD2A (lab raised, see Black et. al Mol cell 2010) bound to magnetic protein A agarose (Invitrogen). The immune complexes were collected by magnet and washed with the following buffers twice each for 5 min at room temperature;  Low salt buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 150 µl elution buffer (1% SDS, 10mM Tris pH 8.0) at 55C with the addition of proteinaseK for 1 hr at 55 degrees C.  After reversal of crosslinks at 65C, genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/10 of the material used for ChIP) by Promega SV wizard DNA purification columns following the manufacturers protocol.  ChIP was validated at chr1Sat2 (see Black et. al Mol cell 2010) by qPCR prior to labeling and hybridization.    Subconfluent 15cm dishes (~5x10^6 cells) were fixed with 1% formaldehyde in culture medium for 15 min at 37 degrees C followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed. The crosslinked chromatin was sheared to an average size of 300 bp using a cup horn at 4 degrees C (Misonix). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (20 µg) was RNAse A treated for 1 hr and diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and was then incubated overnight with anti-JMJD2A (lab raised, see Black et. al Mol cell 2010) bound to magnetic protein A agarose (Invitrogen). The immune complexes were collected by magnet and washed with the following buffers twice each for 5 min at room temperature;  Low salt buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 150 µl elution buffer (1% SDS, 10mM Tris pH 8.0) at 55C with the addition of proteinaseK for 1 hr at 55 degrees C.  After reversal of crosslinks at 65C, genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/10 of the material used for ChIP) by Promega SV wizard DNA purification columns following the manufacturers protocol.  ChIP was validated at chr1Sat2 (see Black et. al Mol cell 2010) by qPCR prior to labeling and hybridization.

value: HEK293T GFP-JMJD2A

source_name: Input DNA from HEK293T cells overexpressing GFP-JMJD2A

value: HEK293T overexpressing GFP-JMJD2A


title: 14286376 - WT + MTX roots Repet2 vs WT roots Repet2

type: RNA

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

country: France

institute: INRA

description: Folates and 1C metabolism

hyb_protocol: WT roots Repet2 Cy5 / WT + MTX roots Repet2 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%

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.

molecule: total RNA

organism: Arabidopsis thaliana

source_name: WT roots Repet2

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) 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: roots - Media=Murashige and Skoog medium (pH 5.7) containing 1% (w/v) Suc, phytagar (0.5%–1%), and vitamins pyridoxine-HCl, nicotinic acid (0.5 mg/mL), and thiamine (1 mg/mL). Plates were incubated vertically in a growth room. For 5-Formyl-THF [(6S)-5-formyl-5,6,7,8-tetrahydrofolic acid, calcium salt; natural calciumfolinate; Schircks Laboratories] and Methotrexate [(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]pentanedioic acid; Schircks Laboratories] supplementation, stock solutions were applied to the growth medium to achieve the desired working concentration. hygrometry- Temperature=19ºCNight-22ºCDay Light=120-140 mmol photons m2/s

extract_protocol: WT roots Repet2:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

treatment_protocol: no treatment

tag: ecotype

value: columbia

tag: genotype/variation

value: wild type

tag: tissue

value: roots

tag: dev.stage (boyes et al. plant cell 2001)

source_name: WT + MTX roots Repet2

extract_protocol: WT + MTX roots Repet2:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

treatment_protocol: Name:Methotrexate supplemented - compound based treatment - compound addition,methotrexate:quantity 5nM time 17day . Arabidopsis thaliana were seeded after cold treatment (3 days) and grown for 17 days in MS media with different treatments in growth room. After this time shoots and roots were collected and separated with a razor blade, flash frozen with liquid nitrogen and stored at -80ºC for further RNA extraction.


title: H4 +N Kb8

city: Charleston

first: Frances

middle: M

last: Van Dolah

state: SC

address: 219 Fort Johnson Rd

institute: Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research

hyb_protocol: 480 ng of Cy3 labeled targets were hybridized to the array for 17 hours at 60°C.  After hybridization, arrays were washed according to the manufacturer’s protocol (Agilent, Santa Clara, CA).

scan_protocol: Microarrays were imaged using an Agilent microarray scanner using extended dynamic range scanning

data_processing: Images were extracted with Agilent Feature Extraction version 9.5.3.1 using a rank consistency filter and a combination linear and LOWESS normalization algorithm.

organism: Karenia brevis

source_name: Karenia brevis, N-replete, newly transcribed RNA during fourth hour post-N-addition

label_protocol: Newly synthesized RNA (25 ng) was amplified and labeled with Cy3 dye using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA).  The amplified, labeled RNA was quantified using a NanoDrop ND-1000.

growth_protocol: Karenia brevis, Wilson isolate, was grown in 1 L batch cultures in autoclaved, 20 µm filtered seawater at 36 ‰ enriched with f/2 medium, modified to contain only 10 µM NO3, at 25 °C  on a 16 h:8 h light:dark cycle with illumination from cool white lights at 150 µE m-2 sec-1

extract_protocol: Cultures were independently harvested by centrifugation.  Total RNA from each sample was prepared using TriReagent.  Following precipitation in ethanol, the RNA was resuspended and purified using a Qiagen RNeasy column.  Total RNA was quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100.  Total RNA was biotinylated with Pierce EZ-link Biotin-HPDP, precipitated in ethanol, and quantified using a NanoDrop ND-1000.  This RNA was then separated into pre-exisiting (non-biotinylated) and newly synthesized (biotinylated) RNA pools using streptavidin coated magnetic beads.  Once separated, the newly synthesized RNA fraction was precipitated with isopropanol and quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100.

treatment_protocol: 155 µM NO3 was added to stationary phase (Day 9) cultures grown in 10 µM NO3 (n=6).  0.2 mM 4-thiouracil in DMSO was added for 1 h during the first (n=3) or fourth (n=3) hour post-N-addition to cultures to biosynthetically label newly synthesized RNA.  Additionally, 0.2 mM 4-thiouracil in DMSO was added for 1 h to stationary phase (Day 9) cultures grown in 10 µM NO3 (n=3).

tag: isolate

value: Wilson Isolate

tag: [no3] added on day 9

tag: 4-thiouracil labeling time

value: fourth h post-N-addition


title: plasma from nasopharyngeal carcinoma patient [51A]

city: GuangZhou

first: jinze

last: zheng

address: Dongfeng east road 651#

country: China

institute: Sun Yat-Sen University Cancer Center

description: MicroRNA expression profiles in plasma

hyb_protocol: The labeling and hybridization procedures were performed according to the protocols in the Agilent microRNA Microarray System.

scan_protocol: The microarray image information was converted into spot intensity values using Feature Extraction Software Rev. 9.5.3 (Agilent Technologies, Santa Clara, CA). The signal after background subtraction was exported directly into the GeneSpring GX10 software (Agilent Technologies, Santa Clara, CA). .

data_processing:
The raw signals obtained for single-color CY3 hybridization were normalized to the endogenous control mir-454.
A log transformation of base 10 was performed before data analysis

label_protocol: Total RNA (100 ng) derived from the plasma samples was labeled with Cy3.

extract_protocol: RNA from 400 μL of plasma was isolated using the mirVana PARIS microRNA Isolation kit according to the manufacturer’s instructions (Ambion, Austin, TX). Total RNA from cells was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA concentration was quantified using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Waltham, MA). 1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

treatment_protocol: For the plasma preparation, peripheral blood (3 ml) was drawn into EDTA tubes. Within 30 min, the tubes were centrifuged at 820 g for 10 min. Then, 1-ml aliquots of the plasma were transferred to 1.5-ml tubes and centrifuged at 16,000 g for another 10 min to remove any remaining cellular debris. Subsequently, the supernatants were transferred to fresh tubes and immediately stored at –80°C.

value: Peripheral Blood

tag: gender

value: male

tag: age

value: 56 years


title: plasma from nasopharyngeal carcinoma patient [42A]

description: MicroRNA expression profiles in plasma

value: 47 years


title: BrdU 2A UBQ ChIP-chip replicate 3

description:
BrdU ChIP-chip in GFP-JMJD2A and ubiquitin overexpressing HEK293T cells
Also included is the Normalized data used to generate Figure 5.txt file.  This file contains the data used to generate the graph to in Figure 5 of Van Rechem et al. JBC 2011.  It is the smoothed, averaged BrdU signal intensities in the 2 megabases surrounding JMJD2A binding sites and randomly selected binding sites.

source_name: BrdU ChIP DNA from HEK293T cells overexpressing GFP-JMJD2Aand Ubiquitin

value: BrdU

tag: chip antibody manufacturer

value: BD Sciences

tag: chip antibody catalog #

tag: chip antibody lot #


title: 14286373 - WT + 5CHO-THF leaves Repet3 vs WT leaves Repet3

description: Folates and 1C metabolism

hyb_protocol: WT leaves Repet3 Cy5 / WT + 5CHO-THF leaves Repet3 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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.

growth_protocol: caulin leaf - Media=Murashige and Skoog medium (pH 5.7) containing 1% (w/v) Suc, phytagar (0.5%–1%), and vitamins pyridoxine-HCl, nicotinic acid (0.5 mg/mL), and thiamine (1 mg/mL). Plates were incubated vertically in a growth room. For 5-Formyl-THF [(6S)-5-formyl-5,6,7,8-tetrahydrofolic acid, calcium salt; natural calciumfolinate; Schircks Laboratories] and Methotrexate [(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]pentanedioic acid; Schircks Laboratories] supplementation, stock solutions were applied to the growth medium to achieve the desired working concentration. hygrometry- Temperature=19ºCNight-22ºCDay Light=120-140 mmol photons m2/s

source_name: WT + 5CHO-THF leaves Repet3

extract_protocol: WT + 5CHO-THF leaves Repet3:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

treatment_protocol: Name:5-Formyl-THF supplemented - compound based treatment - compound addition,5-formyl-thf:quantity 500uM time 17day . Arabidopsis thaliana were seeded after cold treatment (3 days) and grown for 17 days in MS media with different treatments in growth room. After this time shoots and roots were collected and separated with a razor blade, flash frozen with liquid nitrogen and stored at -80ºC for further RNA extraction.


title: plasma from nasopharyngeal carcinoma patient [62A]

description: MicroRNA expression profiles in plasma

value: 40 years


title: BrdU 2A UBQ ChIP-chip replicate 2

value: BrdU ChIP-chip in GFP-JMJD2A and ubiquitin overexpressing HEK293T cells


title: WT_BNFvsAMN_rep2

city: Bremen

first: Barbara

last: Reinhold-Hurek

department: Microbe-Plant-Interactions

country: Germany

institute: University Bremen

description:
Test: total RNA from cells grown on nitrogen-free synthetic medium on N2.
Reference: total RNA from cells grown with combined nitrogen (ammonia) as nitrogen source.
Biological replicate 2 of 3.
302720_rep2

hyb_protocol: The combined Cy3/Cy5-labelled targets were dried in a speed vac and further dissolved in 60 µl DIG Easy hybridization solution (Roche, Mannheim, Germany). 1 µl of sonicated salmon sperm (5 µg/µl) was further added. The transcriptome microarray spotted on epoxysilane-coated Nexterion Slide E (Schott) (CeBiTec, University Bielefeld) contained 70mer oligonucleotide probes for the 3,989 predicted protein-coding genes from Azoarcus sp. strain BH72 in four replicates (Hauberg 2012). Before hybridization, the targets were denatured at 65°C for 10 min. The hybridization of fluorescently labelled cDNA targets to oligonucleotide microarrays was performed at 42°C in a hybridization chamber (ArrayIt, Sunnyvale, CA, USA) in a water bath for 14 to 18 hours. followed by appropriate washing steps as recommended.

scan_protocol: The Cy3- and Cy5-fluorescence was scanned at 532 nm and 635 nm with the GenePix Scanner 4000A (Molecular Devices, Sunnyvale, CA, USA) with a pixel size of 10 µm.The image analysis was performed with the GenePix 4.1 program and the subsequent analyses were carried out with the open-source software Microarray Data Analysis System (MIDAS v2.19) of TM4 (Saeed et al. 2003, http://www.tm4.org/). After scanning, raw data images of the slides that contain information about gene expression levels were obtained by the GenePix software. These images were analyzed by identifying each spot on the array (with the help of an array layout template) with measurements of its fluorescence intensity and the corresponding background intensity.

data_processing:
With the file transformation tool Express Converter v.2.1 (http://www.tm4.org/utilities.html), data files generated from GenePix (.gpr) were converted to TM4 files (.mev) that could be uploaded to the MIDAS platform. Normalization with MIDAS (TM4 suite) was done in order to reduce variability by appropriately adjusting the data. In this study, the LOWESS (locally weighted scattered plot smoothing) normalization was performed using the block mode as set-up parameter as this mode allowed correction of systematic spatial variation between the grids of the array. The normalized data for each spot were exported to Excel and aligned to the corresponding Azoarcus sp. BH72 gene name.
Overall, three independent experiments (biological experiments) were performed. Average expression fold were determined from normalized values of the three sets. Statistical analysis was performed using the intensity values in a one tail paired t-test. Genes with an expression factor ≥ 1.8 and P value ≤ 0.05 were considered as differentially expressed.

organism: Azoarcus sp.

source_name: pure culture of bacteria, grown with combined nitrogen

label_protocol: For transcriptome microarray analysis, 20 µg of total RNA was reverse transcribed with BioScript RT in respective reaction buffer (Bioline) with random hexamers and 4:1 aminomodified-dUTP/dTTP nucleotide mix for 90 min at 42°C. For coupling of fluorescent dyes to the aminoallyl-labelled first strand cDNA, aliquots of Cy3-NHS or Cy5-NHS esters (GE Healthcare) were dissolved in 60 µl of first strand cDNA, mixed and incubated for 90 min at room temperature in the dark. Blocking of all remaining dyes (quenching) was necessary after labelling. This quenching was achieved by adding 4.5 µl hydroxylamine (4 M) to the sample solution, followed by an incubation for 15 min at room temperature. Subsequent cleaning up of labelled cDNA from remaining dyes was performed with the CyScribe GFX Purification Kit (GE Healthcare) according to manufacturer's instructions. The Cy5- and Cy3-labelled cDNA that was used in one hybridization experiment were cleaned up together. The labelled cDNA (60 µl) was stored at -20°C until microarray hybridization (Hauberg et al. 2012).

growth_protocol: Azoarcus sp. strain BH72 was grown in an oxygen-controlled bioreactor (Biostat B; B. Braun Melsungen AG, Melsungen, Germany) at 37°C with stirring at 6000 r.p.m., under microaerobic conditions (0.6% oxygen concentration) either in nitrogen-free SM-medium on N2, or in the same medium supplemented with 10 mM ammonium chloride as N-source (N-replete).

extract_protocol: The cell pellets were re-suspended in a 1:1 mixture of pre-warmed phenol-chloroform-isoamylalcohol (25:24:1, pH 4.7) and NAES solution (50 mM sodium acetate, 10 mM EDTA, 1 % SDS, pH 5.1). This suspension was incubated for 5 min at 65°C in a water bath, followed by 10 min incubation on ice. For phase separation, the mixture was centrifuged for 10 min at 12°C and 8000 x g. The upper phase was transferred and mixed with the same volume of phenol-chloroform-isoamylalcohol. This phenol extraction was repeated three times, followed by an extraction with equal volume of chloroform isoamylalcohol (24:1). RNA precipitation was performed with the same volume of isopropanol for 45 min on ice. The RNA was pelleted by centrifugation (12900 x g, 10 min at 4°C) and salts were removed with 1 ml of Ethanol (70 %). The dried RNA was dissolved in 100 µl 1 x RNAsecure, frozen in liquid nitrogen and kept at -80°C until further processing. Contaminating DNA was removed from RNA preparations by DNase I using Qiagen columns (RNeasy Mini Kit, Qiagen, Hilden, Germany) according to manufacturer's instructions. The DNase I treated RNA was eluted twice in 30 µL of elution buffer, pooled and finally stored in 1x RNA secure in -80°C.

treatment_protocol: The cells were pelleted by centrifugation (6000 rpm, 28°C, 10 min), pellets were frozen in liquid nitrogen and stored at -80°C until RNA isolation.

tag: strain/background

tag: growth condition

value: combined nitrogen

source_name: pure culture of bacteria, grown under nitrogen fixation

value: nitrogen fixation


title: H4 +N Kb5


title: plasma from nasopharyngeal carcinoma patient [36A]

description: MicroRNA expression profiles in plasma

value: female

value: 60 years


title: H4 +N Kb10


title: 14286378 - WT + MTX leaves Repet3 vs WT leaves Repet3

description: Folates and 1C metabolism

hyb_protocol: WT leaves Repet3 Cy5 / WT + MTX leaves Repet3 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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.

value: columbia

value: leaves

value: MTX


title: 14286372 - WT leaves Repet3 vs WT + 5CHO-THF leaves Repet3

description: Folates and 1C metabolism

hyb_protocol: WT + 5CHO-THF leaves Repet3 Cy5 / WT leaves Repet3 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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.

value: wild type


title: 14286374 - WT roots Repet2 vs WT + MTX roots Repet2

description: Folates and 1C metabolism

hyb_protocol: WT + MTX roots Repet2 Cy5 / WT roots Repet2 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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.

value: roots


title: JMJD2A ChIP-chip replicate 2

tag: cell line

tag: cell type

value: HEK293T GFP-JMJD2A

value: HEK293T overexpressing GFP-JMJD2A


title: H1 +N Kb7

source_name: Karenia brevis, N-replete, newly transcribed RNA during first hour post-N-addition

value: Wilson Isolate

value: first h post-N-addition


title: HMEC_50% confluent_0µg/mL_of_ENP_rep2

city: Nancy

first: Bertrand

last: Rihn

address: 5 rue A Lebrun

department: Faculté de Pharmacie

institute: Université de Lorraine

description: Gene expression in HMEC at 50 % of confluence after 24 h in absence of ENP exposure (0 µg/mL)

hyb_protocol: 600 ng of CTP-Cy3-labelled cRNA (specific activity > 10.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 min in a reaction volume of 25 µl containing 25 x Agilent fragmentation buffer and 10 x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2 x Agilent hybridization buffer were added to the fragmentation mixture and hybridized to SurePrint G3 Human GE v2 8x60K (Agilent) for 17 h at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 (Agilent) and 1 min with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief passage in acetonitrile.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides: scan area was 61x21.6 mm, scan resolution 3 µm, dye channel was set to Green, Tiff file dynamic range was 20 bits and Green PMT was set to 100 %.

data_processing: The scanned images were analyzed with Feature Extraction version 10.10.1.1 (Agilent) using default parameters (protocol GE1_1010_Sep10 and Grid: 039494_D_F_20120628) to obtain background subtracted and spatially detrended processed signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: HMEC, 50 % confluent, 0 µg/mL of ENP, replicate 2

label_protocol: CTP-cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Microarray-Based Gene Expression Analysis (Agilent, version 6.6) according to manufacturer's instructions, followed by RNeasy column purification (RNeasy Mini kit, QIAGEN). Dye incorporation and cRNA yield and quality were checked by spectrophotometry (Nanodrop ND1000, Labtech) and with the Agilent 2100 Bioanalyser.

growth_protocol: Human mammary epithelial cells (HMEC 184) is a 10-25 % luminal-like and 75-90 % basal-like breast cell line at prestasis with finite lifespan. It was established and described by Martha R Stampfer (Stampfer MR, Bodnar A, Garbe J, Wong M, Pan A, Villeponteau B, Yaswen P. Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells. Mol Biol Cell. 1997 8:2391-405). Cells were grown in M87A+CT+GFX medium containing (i) 50 % mammary epithelial basal medium (supplemented with 5 mg/ml insulin, 70 µg/ml bovine pituitary extract, 0.5 µg/ml hydrocortisone, 5 ng/ml EGF, 5 µg/ml transferrin, 10-5 M isoproterenol, 2 nM glutamine), (ii) 50 % DMEM/F12 (supplemented with 10 µg/ml insulin, 10 nM tri-iodothyronine, 1 nM β-estradiol, 0.1 µg/ml hydrocortisone, 0.5 % fetal calf serum, 5 ng/ml EGF, 2 mM glutamine, and 1 ng/ml cholera toxin), with (iii) 0.1 nM oxytocin and 0.1 % lipid-rich bovine serum albumin (albuMaxI). Cells were grown in 5 % CO2 atmosphere at 37°C.

extract_protocol: The quality of the RNA extracted with RiboPure kit (Ambion, Austin, TX) was determined by spectrophotometry (A260nm/A280nm > 1.8, BioSpec-nano, Shimadzu) and capillary electrophoresis using RNA 6000 Nano (RIN > 7, Agilent 2100 Bioanalyser™).

treatment_protocol: HMEC 184 cells (50% confluence) were incubated with polymeric Eudragit RS nanoparticles (ENP; 25 µg/mL) for 24h, and without incubation (control) in a T.C. incubator at 37°C and 5% CO2.

value: mammary epithelial cells

tag: treatment

value: control

value: HMEC 184


title: plasma from nasopharyngeal carcinoma patient [140B]

description: MicroRNA expression profiles in plasma

value: Peripheral Blood

value: male

value: 44 years


title: BrdU CTRL ChIP-chip replicate 2

description:
BrdU ChIP-chip in GFP  overexpressing HEK293T cells
Also included is the Normalized data used to generate Figure 5.txt file.  This file contains the data used to generate the graph to in Figure 5 of Van Rechem et al. JBC 2011.  It is the smoothed, averaged BrdU signal intensities in the 2 megabases surrounding JMJD2A binding sites and randomly selected binding sites.

source_name: BrdU ChIP DNA from HEK293T cells overexpressing GFP

value: HEK293T GFP

tag: chip antibody


title: BrdU 2A UBQ ChIP-chip replicate 1

value: BrdU

value: BD Sciences

value: BrdU ChIP-chip in GFP-JMJD2A and ubiquitin overexpressing HEK293T cells


title: plasma from nasopharyngeal carcinoma patient [131B]

description: MicroRNA expression profiles in plasma

value: 38 years


title: H1 +N Kb11


title: BrdU 2A ChIP-chip replicate 1

description:
BrdU ChIP-chip in GFP-JMJD2A overexpressing HEK293T cells
Also included is the Normalized data used to generate Figure 5.txt file.  This file contains the data used to generate the graph to in Figure 5 of Van Rechem et al. JBC 2011.  It is the smoothed, averaged BrdU signal intensities in the 2 megabases surrounding JMJD2A binding sites and randomly selected binding sites.

source_name: BrdU ChIP DNA from HEK293T cells overexpressing GFP-JMJD2A


title: PHF6shRNA-2, biological duplicate 1

city: Boston

first: Chi

last: Zhang

address: 107 Avenue Louis Pasteur

institute: Harvard Medical School

hyb_protocol: whole transcript sense target was generated using Ambion WT expression kit and Affymetrix GeneChip WT terminal labeling hybridization kit

scan_protocol: processed using Affymetrix GeneAtlas system according to manufacturer instructions

data_processing: The data were analyzed with customized scripts. Raw data were background-corrected and normalized using the RMA algorithm (Affymetrix PowerTools). Data were filtered for unannotated probes, for genes with no expression in any condition (EV < 120), and for probes giving discordant data between replicates (interreplicate CV > 0.7).

label: biotin

organism: Rattus norvegicus

source_name: PHF6 KO_cortical neurons

growth_protocol: primary cultured rat cortical neuron

extract_protocol: Trizol extraction was performed according to manufacturer's instruction

treatment_protocol: neurons are cultured in standard conditions. No treatment other than lentivirus infection is included

tag: strain background

value: sprague dawley

tag: developmental stage

value: cortical neurons

tag: infected with

value: pLL3.7 lentivirus expressing PHF6shRNA-2

tag: genotype/variation

value: PHF6 knockdown

tag: time point

value: 5 days after infection


title: PHF6shRNA-2, biological duplicate 2

label_protocol: whole transcript sense target was generated using Ambion WT expression kit and Affymetrix GeneChip WT terminal labeling hybridization kit

tag: tissue


title: plasma from nasopharyngeal carcinoma patient [129B]

description: MicroRNA expression profiles in plasma

tag: gender

value: female

value: 27 years


title: H1 +N Kb1

tag: isolate

value: Wilson Isolate

tag: [no3] added on day 9

tag: 4-thiouracil labeling time

value: first h post-N-addition


title: plasma from nasopharyngeal carcinoma patient [16A]

description: MicroRNA expression profiles in plasma

value: Peripheral Blood

tag: age

value: 39 years


title: plasma from nasopharyngeal carcinoma patient [127B]

description: MicroRNA expression profiles in plasma

value: male

value: 42 years


title: BrdU CTRL UBQ ChIP-chip replicate 3

value: HEK293T GFP

value: BrdU ChIP-chip in GFP and ubiquitin  overexpressing HEK293T cells


title: plasma from nasopharyngeal carcinoma patient [5A]

description: MicroRNA expression profiles in plasma

value: 43 years


title: plasma from nasopharyngeal carcinoma patient [123A]

description: MicroRNA expression profiles in plasma

value: 56 years


title: BrdU 2A ChIP-chip replicate 2

value: HEK293T GFP-JMJD2A

value: BrdU

tag: chip antibody manufacturer

value: BD Sciences

tag: chip antibody catalog #

tag: chip antibody lot #

value: BrdU ChIP-chip in GFP-JMJD2A overexpressing HEK293T cells


title: plasma from nasopharyngeal carcinoma patient [114B]

description: MicroRNA expression profiles in plasma

value: 41 years


title: plasma from nasopharyngeal carcinoma patient [108B]

description: MicroRNA expression profiles in plasma

value: female

value: 62 years


title: control, biological duplicate 1

source_name: wt_cortical neurons

value: sprague dawley

value: cortical neurons

value: pLL3.7 lentivirus expressing control shRNA

value: wild type

value: 5 days after infection


title: WT_BNFvsAMN_rep3

description:
Test: total RNA from cells grown on nitrogen-free synthetic medium on N2.
Reference: total RNA from cells grown with combined nitrogen (ammonia) as nitrogen source.
Biological replicate 3 of 3.
302724_rep3

tag: strain/background

tag: growth condition

value: nitrogen fixation

value: combined nitrogen


title: 1H -N Kb3

source_name: Karenia brevis, N- limited, newly transcribed RNA during 1 h

value: Wilson Isolate

value: 0 µM

value: 1 h


title: 1H -N Kb12


title: plasma from nasopharyngeal carcinoma patient [105B]

description: MicroRNA expression profiles in plasma

value: Peripheral Blood

value: male

value: 49 years


title: New RNA Kb6

source_name: Karenia brevis, newly synthesized RNA

label_protocol: Total RNA (100 ng) , pre-existing RNA (100 ng), and newly synthesized RNA (25 ng) were independently amplified and labeled with Cy3 dye using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA).  The amplified, labeled RNA was quantified using a NanoDrop ND-1000.

growth_protocol: Karenia brevis, Wilson isolate, was grown in 1 L batch cultures in autoclaved, 20 µm filtered seawater at 36 ‰ enriched with f/2 medium at 25 °C  on a 16 h:8 h light:dark cycle with illumination from cool white lights at 150 µE m-2 sec-1

extract_protocol: Six replicate cultures were independently harvested by centrifugation.  Total RNA from each sample was prepared using TriReagent.  Following precipitation in ethanol, the RNA was resuspended and purified using a Qiagen RNeasy column.  Total RNA was quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100.  Total RNA was biotinylated with Pierce EZ-link Biotin-HPDP, precipitated in ethanol, and quantified using a NanoDrop ND-1000.  This RNA was then separated into pre-exisiting (non-biotinylated) and newly synthesized (biotinylated) RNA pools using streptavidin coated magnetic beads.  Once separated, each fraction was precipitated with isopropanol and quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100.

treatment_protocol: 0.2 mM 4-thiouracil in DMSO was added to cultures to biosynthetically label newly synthesized RNA.  Cultures were harvested 2 h post 4-thiouracil addition

tag: rna pool

value: newly synthesized RNA


title: Pre-Existing RNA Kb6

source_name: Karenia brevis, pre-existing RNA

value: pre-existing RNA


title: plasma from nasopharyngeal carcinoma patient [312]

description: MicroRNA expression profiles in plasma

value: female

value: 45 years


title: BrdU 2A ChIP-chip replicate 3

tag: cell type

value: HEK293T GFP-JMJD2A

tag: cell line

tag: chip antibody

value: BrdU

value: BD Sciences

tag: treatment

value: BrdU ChIP-chip in GFP-JMJD2A overexpressing HEK293T cells


title: BrdU CTRL ChIP-chip replicate 1

value: HEK293T GFP

value: BrdU ChIP-chip in GFP  overexpressing HEK293T cells


title: plasma from nasopharyngeal carcinoma patient [230]

description: MicroRNA expression profiles in plasma


title: plasma from nasopharyngeal carcinoma patient [243]

description: MicroRNA expression profiles in plasma

value: 38 years


title: New RNA Kb5

value: Wilson Isolate


title: Total RNA Kb6

source_name: Karenia brevis, total RNA

value: total RNA


title: plasma from nasopharyngeal carcinoma patient [221]

description: MicroRNA expression profiles in plasma

value: 55 years


title: plasma from nasopharyngeal carcinoma patient [195]

description: MicroRNA expression profiles in plasma

value: Peripheral Blood

value: 42 years


title: plasma from nasopharyngeal carcinoma patient [198]

description: MicroRNA expression profiles in plasma

value: 56 years


title: Total RNA Kb5


title: New RNA Kb4

value: newly synthesized RNA


title: WT_BNFvsAMN_rep1

description:
Test: total RNA from cells grown on nitrogen-free synthetic medium on N2.
Reference: total RNA from cells grown with combined nitrogen (ammonia) as nitrogen source.
Biological replicate 1 of 3.
302717_rep1

tag: genotype/variation

value: wild type

value: combined nitrogen

value: nitrogen fixation


title: plasma from nasopharyngeal carcinoma patient [177]

description: MicroRNA expression profiles in plasma

tag: tissue

value: male

value: 37 years


title: plasma from nasopharyngeal carcinoma patient [178]

description: MicroRNA expression profiles in plasma

tag: gender

value: female

value: 45 years


title: Pre-Existing RNA Kb4

value: pre-existing RNA

tag: isolate


title: Total RNA Kb4


title: plasma from nasopharyngeal carcinoma patient [175]

description: MicroRNA expression profiles in plasma

value: 48 years


title: plasma from nasopharyngeal carcinoma patient [174]

description: MicroRNA expression profiles in plasma


title: plasma from nasopharyngeal carcinoma patient [170]

description: MicroRNA expression profiles in plasma


title: BrdU CTRL UBQ ChIP-chip replicate 1

source_name: BrdU ChIP DNA from HEK293T cells overexpressing GFP and Ubiquitin

value: HEK293T GFP

value: BrdU ChIP-chip in GFP and ubiquitin  overexpressing HEK293T cells


title: Pre-Existing RNA Kb3

value: Wilson Isolate


title: plasma from nasopharyngeal carcinoma patient [162]

description: MicroRNA expression profiles in plasma


title: New RNA Kb3


title: Array 2_2: SUS_2 cy3+PYR_2 cy5

city: Harpenden

first: Chris

last: Bass

state: Hertfordshire

address: West Common

department: Biological Chemistry

country: United Kingdom

institute: Rothamsted Research

hyb_protocol: See above

scan_protocol: Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version 10.5).

data_processing: Agilent Feature Extraction Software (v 10.5) was used for background subtraction and LOWESS normalization.

organism: Spodoptera frugiperda

source_name: five L2 larvae

label_protocol: Two micrograms of each RNA sample was used to generate labeled cRNA, this was prepared, hybridized onto arrays and these were washed and scanned exactly as described in the Agilent Quick Amp Labeling Protocol (Version 5.7) which can be downloaded from http://www.chem.agilent.com/en-US/products/instruments/dnamicroarrays/pages/gp11617.aspx.

growth_protocol: All strains were maintained on artificial diet at 25± 1°C with a 16:8 L:D photoperiod

extract_protocol: Total RNA extracted using Trizol following manufacturer's instructions. Genomic DNA was removed by DNase I digestion using the DNA-free DNase Treatment and Removal Reagent (Ambion).

treatment_protocol: OP and PYR strains were maintained in the laboratory under selection with chlorpyrifos (at increasing discriminating doses from 100 up to 400 µg of insecticide/g of insect in a topical bioassay) or lambda-cyhalothrin (from  8.4 up to 27 µg of insecticide/g of insect) respectively

tag: clone

value: SUS

value: PYR


title: Array 2_1: SUS_1 cy5+PYR_1 cy3


title: Array 2_3: SUS_3 cy5+PYR_3 cy3


title: Array 1-4: SUS_4 cy3 + OP_4 cy5

value: OP


title: CM_TRAP_7dpa-3

city: Durham

first: Kenneth

middle: Daniel

last: Poss

state: NC

address: 349 Nanaline Duke Building, Box 3709,  Duke University Medical Center

department: Cell Biology

institute: Duke University, HHMI

description: This sample is the third of the three 7 dpa biological replicates (cmlc2:TRAP)used in the experiments.

hyb_protocol: Hybridization was performed by Mogene, LC (St. Louise, MO), following their standard operating protocol. See www.nimblegen.com.

scan_protocol: Scanning was performed by Mogene, LC (St. Louise, MO), following their standard operating protocol. See www.nimblegen.com.

data_processing: The raw data(.pair file) was subjected to RMA (Robust-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package.

organism: Danio rerio

source_name: 7 dpa zebrafish cmlc2:TRAP line

label_protocol: Labeling was performed by Mogene, LC (St. Louise, MO), following their standard operating protocol. See www.nimblegen.com.

growth_protocol: Zebrafihs (of EkkWill strain) at 5-6 months of age were used for ventricular resection and ribosome-associated RNA isolation. Cmlc2:TRAP transgenic fish were analyzed as hemizogotes.

extract_protocol: 50 apical halves of ventricles from cmlc2:TRAP zebrafish were collected, homogenized and subjected to polysome purification as described previously (Heiman M, et al., Cell 2008).  RNAs were purified with Picopure RNA isolation kit (Arcturus), and cDNAs were synthesized and amplified using the Nugen Pico WTA system (Nugen, CA).

treatment_protocol: Zebrafish were anaesthetized in Tricaine, and a small incision penetrating the skin and pericardial sac was made to expose the ventricle. After removal of 20% ventricle at the apex, fish were returned to water and stimulated to breathe.  Then, zebrafish were allowed to grow for 1 and 7 days before sarcrifice for RNA isolation.

value: Heart

tag: time

value: 7 dpa

tag: genotype

value: cmlc2:TRAP line

tag: sample type

value: Ribosome-associated RNA

tag: strain

value: EkkWill

value: injured heart


title: Array 2-4: SUS_4 cy3+PYR_4 cy5


title: Array 1_2: SUS_2 cy3 + OP_2 cy5


title: Array 1_3: SUS_3 cy5 + OP_3 cy3


title: WH8102_MC-shock_replicate 3

city: Sydney

first: Sasha

last: Tetu

state: NSW

address: North Ryde

department: CMBS

country: Australia

institute: Macquarie University

hyb_protocol: Microarray slides were immersed in pre-hybridization buffer (1 % BSA, 5X SSC, 0.1 % SDS, 0.45 mm-filtered) before hybridization. Slides were washed with sterile water (4x) and isopropanol (3x), spin-dried and covered with lifterslip. cDNA probes were dried using Speedvac and then resuspended in hybridization buffer (50 % formamide, 5X SSC, 0.1 % SDS, 0.45 mm-filtered). Probes were heated at 95 °C (15 min) before applying to the slides. Hybridization was performed overnight at 42°C.

scan_protocol: Slides were scanned at 10-um resolution using an Axon 4000B scanner with GenePix 4.0 software

data_processing: Processing of the TIFF images from hybridized arrays was performed using TIGR-Spotfinder (www.tigr.org/software), and the datasets normalized by applying the LOWESS algorithm, using block mode and a smooth parameter of 0.33, available in the TIGR-MIDAS package (www.tigr.org/software). Statistical analysis was performed on the mean of log2-transformed signal ratios of the replicate spots using the Statistical Analysis of Microarrays (SAM) algorithms  with a false discovery rate of less than 1%.

organism: Parasynechococcus marenigrum WH 8102

source_name: Synechococcus sp. WH8102, no treatment

label_protocol: Reverse transcription of RNA samples was performed using the SuperScript Plus Indirect cDNA Labeling System (Invitrogen) with random hexamer primers.  Approximately 4 µg of total RNA was used for indirect labelling, leading to the production of approximately 4 µg of cDNA with approximately 200 pmol of dye molecule incorporated per microgram of cDNA synthesized.

growth_protocol: Axenic Synechococcus sp. WH8102 and Synechococcus sp. CC9311 cultures were maintained in either SN medium or in an artificial seawater medium (SOW) with 9.0 mM NaNO3 standard.  For growth assays, batch cultures were grown in glass flasks, gently stirred, or in glass tubes, at 25 °C under 30 μEinstein m-2 s-1 continuous white light.

extract_protocol: Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions, RNA was resuspended in 100 μl of DEPC-treated water, further processed using RNeasy Mini kit (Qiagen) following the manufacturer’s protocol, twice digested with DNase I (Qiagen), then eluted from the columns.

treatment_protocol: Duplicate 1.5-liter batch cultures of WH8102 and CC9311 were grown up to the early exponential phase and half the culture was spun down at room temperature, resuspended in Trizol reagent (Invitrogen), and frozen at -80°C for RNA extraction in parallel with treated cells, taking care that the harvesting time stayed under 30 min. EB (final concentration 2 μg/mL) or MC (final concentration 0.5 μg/mL) were added to the remaining culture. After a two hour incubation, these cells were harvested and suspended in Trizol reagent as above.

value: no treatment

source_name: Synechococcus sp. WH8102, MC treated

value: MC treated


title: WH8102_MC-shock_replicate 4


title: WH8102_MC-shock_replicate 5


title: WH8102_MC-shock_replicate 6


title: WH8102_EB-shock_replicate 6

source_name: Synechococcus sp. WH8102, EB treated

value: EB treated


title: WH8102_MC-shock_replicate 2


title: WH8102_EB-shock_replicate 5


title: CM_TRAP_7dpa-1

description: This sample is the first of the three 7 dpa biological replicates (cmlc2:TRAP)used in the experiments.


title: CM_TRAP_7dpa-2

description: This sample is the second of the three 7 dpa biological replicates (cmlc2:TRAP)used in the experiments.


title: CM_TRAP_1dpa-3

description: This sample is the third of the three 1 dpa biological replicates (cmlc2:TRAP)used in the experiments.

source_name: 1 dpa zebrafish cmlc2:TRAP line

value: 1 dpa


title: CM_TRAP_1dpa-1

description: This sample is the first of the three 1 dpa biological replicates (cmlc2:TRAP)used in the experiments.


title: CM_TRAP_uninjured1

description: This sample is the first of the three uninured biological replicates (cmlc2:TRAP) used in the experiments.

source_name: uninjured zebrafish cmlc2:TRAP line

value: control

value: uninjured heart


title: CM_TRAP_uninjured3

description: This sample is the third of the three uninured biological replicates (cmlc2:TRAP)used in the experiments.


title: Array 1_1: SUS_1 cy5 + OP_1 cy3


title: CM_TRAP_1dpa-2

description: This sample is the second of the three 1 dpa biological replicates (cmlc2:TRAP)used in the experiments.


title: CM_TRAP_uninjured2

description: This sample is the second of the three uninured biological replicates (cmlc2:TRAP)used in the experiments.


title: WH8102_EB-shock_replicate 3


title: BrdU CTRL ChIP-chip replicate 3

value: HEK293T GFP

value: BrdU ChIP-chip in GFP  overexpressing HEK293T cells


title: WH8102_EB-shock_replicate 2


title: WH8102_EB-shock_replicate 1


title: WH8102_EB-shock_replicate 4


title: W_NC_HTB46_0901808

status: Public on Jul 31 2013

city: Grand Rapids

first: Karl

last: Dykema

state: MI

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

description: Gene expression data were obtained from 786-O and Caki-1 cell lines.

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome U133 Plus 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

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

data_processing: The data were analyzed with MBNI re-mapped CDF files, version 12. RMA normalization was performed.

label_protocol: 10 ug of Qiagen-purified total RNA. R-Phycoerythrin Streptavidin (SAPE), from Molecular Probes, 1 mg/ml (we use 6 ul per reaction).

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

value: renal cell carcinoma (RCC)

tag: subtype


title: 1H -N Kb6

value: Wilson Isolate

tag: [no3] added on day 9

value: 0 µM

tag: 4-thiouracil labeling time

value: 1 h


title: t_070208


title: nc_070208


title: CHOPWT2.1 (DK_A11_11)

status: Public on Jul 26 2013

city: Philadelphia

first: Jason

last: Mills

state: PA

address: 3501 Civic Center Blvd, CTRB, RM5100

department: Laboratory Medicine

institute: The Children's Hospital of Philadelphia

hyb_protocol: Three biological replicates were prepared for all iPSC and hESC-derived samples. Quantified RNA samples were hybridized to Affymetrix GeneChip Human Gene 1.0 ST arrays.

scan_protocol: Affymetrix protocol

data_processing: All gene array data obtained from Affymetrix passed quality control steps and were sent to RMA normalization and log transformation by Affymetrix Expression Console.

source_name: wild type hiPS

label_protocol: Affymetrix protocol

growth_protocol: Human fibroblast lines CHOPWT1 (Coriell GM00969), CHOPWT2 (Coriell GM05565), and BMC (ATCC CRL-2097) were cultured in IMDM (Mediatech) containing 10% FBS at 37°C and 5% CO2 in a humidified incubator. The iPSCs were maintained in HES media: DMEM/F12 containing 2 mM of glutamine, 20% Knockout Serum Replacement (KO), 1X NEAA, 0.1 mM β-mercaptoethanol, and 10 ng/ml of bFGF (Roche, Indianapolis, IN) on 0.1% gelatin (Sigma, Inc.) coated plates with irradiated mouse embryonic fibroblast (MEFs)

extract_protocol: Total RNA and miRNA were isolated from cells using the miRNeasy kit (Qiagen, Inc), with the optional column RNAse-free DNase treatment, according to the manufacturer’s instructions.

treatment_protocol: iPSCs were enriched by FACS sorted for pluripotency markers SSEA3-FITC (1:100; Biolegend) and Tra-1-81-APC (1:100; Biolegend)

value: Induced pluripotent stem cells


title: CHOPWT2.2 (DK_B2_14)


title: CHOPWT2.2 (DK_B1_13)


title: CHOPWT2.2 (DK_B3_15)


title: CHOPWT2.1 (DK_A12_12)


title: CHOPWT2.1 (DK_A10_10)


title: HIV-1 Tat SH-SY5Y (miRNA)

first: Bassel

middle: E

last: Sawaya

address: 3307 N. Broad Street

department: Neurology

institute: Temple University

hyb_protocol: RNA was labeled using the miRCURY LNATM miRNA, Hy5 Power Labeling Kit (Exiqon) as per manufacturer’s recommendations using Maui SC Hybridization Chambers (BioMicro Systems, Salt Lake City, UT).

scan_protocol: Array chips were scanned using Axon GenePix Scanner (Molecular Devices, Downingtown, PA) and Genepix 4000 image capture software (Molecular Devices, Downingtown, PA).

data_processing: Quantile normalization was used using JMP Genomics (JMP, Cary, NC) at Harvard Catalyst - Laboratory for Innovative Translational Technology (HC-LITT) and differentially regulated miRNAs were determined. Untreated cells were used as control.

label: Hy5 Power labeling

source_name: Human Neuronal Cell line

growth_protocol: Neuronal cell line, SH-SY5Y (5 x 105) were grown in DMEM + 10% FBS on MatTek glass bottom plates treated with collagen (MatTek, Ashland, MA).

extract_protocol: Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA). 750 ng of total RNA was used for miRNA array analysis using miRCURY LNATM microRNA Array, v. 11.0 (Exiqon, Woburn, MA).

treatment_protocol: Cells were treated with Tat (10 ng/ml) kindly received from NIH (AIDS reagnets Bank) for 24 hours.

value: Tat (10 ng/ml)


title: CHOPWT2.3 (DK_B4_16)


title: CHOPWT1.2 (DK_A5_5)


title: CHOPWT2.3 (DK_B6_18)


title: CHOPWT1.2 (DK_A4_4)


title: CHOPWT1.2 (DK_A6_6)


title: CHOPWT1.1 (DK_A2_2)


title: CHOPWT1.1 (DK_A3_3)


title: CHOPWT1.3 (DK_A7_7)


title: CHOPWT1.3 (DK_A9_9)


title: CHOPWT2.3 (DK_B5_17)


title: CHOPWT1.3 (DK_A8_8)


title: Wt vs delta esl1/ delta esl2 (rep 3)

status: Public on Jul 18 2013

city: Clayton

first: traude

middle: Helene

last: Beilharz

state: Victoria

address: Monash University

department: Biochemistry & Molecular Biology

institute: Monash University

description: Biological replicate 3 of 3.

hyb_protocol: 300ng of Cy3 labelled cRNA and 300ng of Cy5 labelled cRNA were co-hybridised to Agilent 8 x 15K custom arrays. Arrays were hybridised at 65C for 17 hours. Arrays were washed according to manufacture's instructions.

scan_protocol: Arrays were scanned on an Agilent G2565CA Scanner at a 5um resolution. Data was extracted from the image files using Agilent Feature Extraction Software 9.5.3 using protocol GE2 QCMT Feb07

data_processing: Lowess normalisation (gene-spring)

organism: Saccharomyces cerevisiae

source_name: Yeast growing at log phase in rich media

label_protocol: 500ng of total RNA was labelled using T7 polymerase (Agilent Quick Amp Two Colour Labeling kit protocol version 5.7, March 2008). Agilent RNA spikes were included to monitor labelling

growth_protocol: Experiments were carried out in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C. Yeast cells were harvested during log phase

extract_protocol: RNA was extracted using the hot-phenol method

value: Y52 strain

value: MATa ade2-1 can1-100 leu2-3, 122 trp1-1 ura3-1 RAD5/esl1∆::KAN esl2∆::NAT


title: CHOPWT1.1 (DK_A1_1)


title: Wt vs delta esl1/ delta esl2 (rep 3) Dye-Swap

description: Dye swap of replicate  3.

value: MATa ade2-1 can1-100 leu2-3, 122 trp1-1 ura3-1 RAD5


title: W_T_HTB46_0901808


title: Wt vs delta esl1/ delta esl2 (rep 2)

description: Biological replicate 2 of 3.


title: Mock SH-SY5Y (miRNA)

source_name: SH-SY5Y neuronal cell line

label_protocol: RNA was labeled using the miRCURY LNATM miRNA, Hy5 Power Labeling Kit (Exiqon) as per manufacturer’s recommendations using Maui SC Hybridization Chambers (BioMicro Systems, Salt Lake City, UT).

treatment_protocol: Cells were treated with 10 pg/ml of Vpr protein kindly received from Dr Eric Cohen, University of Montreal-Canada for 24 hours.


title: Pre-Existing RNA Kb5

tag: rna pool

value: pre-existing RNA


title: control, biological duplicate 2

source_name: wt_cortical neurons

tag: strain background

value: sprague dawley

tag: developmental stage

value: cortical neurons

tag: infected with

value: pLL3.7 lentivirus expressing control shRNA

value: wild type

tag: time point

value: 5 days after infection


title: Wt vs delta esl1/ delta esl2 (rep 1)

description: Biological replicate 1 of 3.

tag: genotype/variation


title: WS iPSC from AG00780 (clone 1.9) rep1

status: Public on Jul 11 2013

city: Bethesda

first: Hoi Hung

last: Cheung

state: MD

address: 49 Convent Dr, Rm.2C08 Bldg.49

institute: NICHD

description: Gene expression data from WS iPSC culture

hyb_protocol: cDNA was hybridization at 45C overnight. Wash and staining was performed by GeneChip Fluidics Station 450 (Affymetrix)

scan_protocol: Arrays were scanned by GeneChip Scanner 3000 7G controlled AGCC Scan Control (Affymetrix)

data_processing: Microarray data were analyzed by Partek Genomics Suite 6.6 with quantile normalization and RMA background correction. A batch effect due to scan date was removed before One-way ANOVA analysis for differentially expressed genes between normal and WS fibroblasts.

source_name: WS iPSC

label_protocol: cDNA was labeled by biotin using the WT Terminal Labeling Kit (Affymetrix).

growth_protocol: Fibroblast culture was maintained in alpha MEM medium containing 15% FBS. iPSC/hESC culture was maintained on Matrigel with mTeSR1 medium.

extract_protocol: Total RNA was extracted by Trizol and purified by Direct-zol RNA MiniPrep (Zymo Research). RNA integrity was assessed by BioAnalyzer.

value: Male

tag: age

value: WRN mutant

tag: cell type

value: Werner Syndrome iPSC


title: WS iPSC from AG12797 rep2

tag: gender


title: WS iPSC from AG00780 (clone 1.9) rep2


title: WS iPSC from AG12797 rep1


title: WS iPSC from AG00780 (clone 2.10) rep1


title: WS iPSC from AG05229 rep1


title: WS iPSC from AG06300 rep1

value: WT


title: WS iPSC from AG06300 rep2


title: WS iPSC from AG00780 (clone 2.10) rep2


title: U2OScells_StablyExpressingBrd4shRNA_rep2

status: Public on Jul 14 2013

city: Cambridge

first: Charles

middle: Arthur

last: Whittaker

institute: Koch Institute

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip HG-U133A_2. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000.

data_processing: R version 2.11.1 (x86_64-pc-linux-gnu), affyPLM_1.24.1, preprocessCore_1.10.0, gcrma_2.20.0, affy_1.26.1, Biobase_2.8.0

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA according to the Affymetrix Expression Analysis Technical Manual.

growth_protocol: 3x10^6 U2OS cells (ATCC) were plated in a 15 cM dish in media containing 10% FBS

extract_protocol: RNA was DNAseI treated and purified using the QIAgen Rneasy mini columns

treatment_protocol: Cells were infected with retroviruses containing hairpins directed against  Brd4 using the mir30-based pMLP vector with primer 5’-TGC TGT TGA CAG TGA GCG AAG ACA CA-3’, control shRNA was in the identical vector with an inactive sequence hairpin with no known target. Stable cell lines expressing the hairpins were then selected with puromycin.

tag: cell line


title: 315a

status: Public on Jun 28 2013

city: Odense C

first: Martin

middle: Jakob

last: Larsen

address: Sdr. Boulevard 29

department: Dept. of Clinical Genetics

country: Denmark

institute: Odense University Hospital

hyb_protocol: Hybridization and washing were performed according to manufacture’s recommendations in a low ozone environment.

scan_protocol: Scanned using a Agilent G2565CA Microarray scanner

data_processing: Scanned images were quantified using Agilent Feature Extraction Software (version 10.7.3.1). Bad quality features flagged during feature extraction were removed and the remaining data were pre-processed. Data were background corrected (normexp, offset=50), then within-array normalized by loess normalization method and between-array normalizatized by the quantile method. The normalized values were used to calculate log2 transformed Cy5/Cy3 ratios. Replicate probes were collapsed by calculating the median. Probes without gene symbol annotation were filtered out. In cases of multiple probes per gene symbol only the probe with the maximum mean (Cy5) intensity was kept. Missing expression values were imputed by k-nearest neighbors averaging (k = 10).

source_name: Breast tumor tissue

label_protocol: RNA was amplified and labeled using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion) according to the manufacturer’s protocol.

extract_protocol: Total RNA was extracted from freshly frozen tumor tissue using Trizol Reagent (Invitrogen) and RNeasy Micro Kit (Qiagen).

value: Premenopausal

tag: who

value: Invasive ductal carcinoma

tag: grade

tag: group

value: Sporadic

value: LumB

tag: er

tag: pr

source_name: Universal Human Reference RNA (Stratagene)

tag: reference


title: 316a

value: Postmenopausal

value: Invasive lobular carcinoma

value: LumA

value: Universal Human Reference RNA (Stratagene)


title: Normal iPSC from GM03440 rep2

description: Gene expression data from normal iPSC culture

source_name: Normal iPSC

tag: genotype

value: Normal iPSC


title: 308a


title: U2OScells_StablyExpressingControlshRNA_rep1

source_name: U2OS cells Expressing Control shRNA


title: 309a

value: Perimenopausal


title: Normal iPSC from GM03440 rep1


title: 241a


title: WH8102_MC-shock_replicate 1

tag: strain

tag: treatment

value: MC treated

value: no treatment


title: 307a

value: BRCA2 c.5576_5579delTTAA, Exon11, p.(Ile1859Lysfs*3)


title: 311a


title: 320a


title: 016a


title: U2OScells_StablyExpressingBrd4shRNA_rep1


title: 305a

value: Mucinous carcinoma


title: WS iPSC from AG05229 rep2

value: Male

value: WRN mutant


title: 304a


title: 297a


title: 302a


title: 286a


title: 293a


title: 291a


title: 285a

value: Tubular carcinoma


title: 287a


title: 300a

value: Premenopausal

value: Basal

value: BRCA1 c.5089T>C, Exon18, p.(Cys1697Arg)


title: 281a

value: Sporadic


title: 283a

value: Postmenopausal

value: Invasive ductal carcinoma


title: 284a

value: LumB


title: 313a

value: Universal Human Reference RNA (Stratagene)


title: 279a

value: LumA


title: 278a


title: 276a


title: 274a


title: 269a

value: BRCA1 c.1556delA, Exon11, p.(Lys519Argfs*13)


title: 266a

value: BRCA2 c.1310_1313delAAGA, Exon10, p.(Lys437Ilefs*22)


title: 255a

value: BRCA2 c.2808_2811delACAA, Exon11, p.(Ala938Profs*21)


title: 262a

value: BRCA1 c.5266dupC, Exon20, p.(Gln1756Profs*74)


title: 247a

value: Medullary carcinoma


title: 273a


title: 264a


title: 243a


title: 240a

value: Other


title: 237a


title: 254a


title: 248a


title: 251a


title: 267a

value: BRCA2 c.8575delC, Exon20, p.(Gln2859Lysfs*4)


title: 231a

value: BRCA1 c.5213G>A, Exon20, p.(Gly1738Glu)


title: 238a

value: Premenopausal

value: Invasive lobular carcinoma


title: 233a


title: 265a

value: Basal


title: 252a

value: Sporadic


title: 242a

value: BRCA1 c.2476delA, Exon11, p.(Thr826Glnfs*20)


title: 246a

value: Postmenopausal

value: Invasive ductal carcinoma


title: 234a

value: LumB

value: Universal Human Reference RNA (Stratagene)


title: 230a


title: 256a


title: 263a

value: LumA


title: 229a


title: 330a


title: 327a

value: BRCA2 c.1813delA, Exon10, p.(Ile605Tyrfs*9)


title: 323a


title: 223a


title: U2OScells_StablyExpressingControlshRNA_rep2


title: 332a


title: 221a


title: 218a


title: 220a


title: 217a


title: 212a

value: BRCA1 c.181T>G, Exon5, p.(Cys61Gly)


title: 216a

value: BRCA1 c.2475delC, Exon11, p.(Asp825Glufs*21)


title: 206a

value: Perimenopausal

value: BRCA2 c.3530_3533delACAG, Exon11, p.(Asp1177Alafs*19)


title: 197a


title: 210a

value: BRCA2 c.6490_6492delinsGACT, Exon11, p.(Gln2164Aspfs*12)


title: 213a


title: 194a

value: BRCA2 c.9015delA, Exon23, p.(Arg3005Serfs*23)


title: 203a

value: BRCA2 c.8575delC, Exon20, p.(Gln2859Lysfs*4)


title: 205a


title: 198a

value: Premenopausal


title: 181a


title: 173a

value: BRCA1 c.115T>G, Exon3, p.(Cys39Gly)


title: 186a

value: Basal


title: 189a

value: Other

value: Invasive lobular carcinoma

value: Sporadic


title: 187a

value: Tubular carcinoma


title: Intellectual_Disability_PN11-1770_2.7M

city: Nijmegen

first: Joep

last: de Ligt

address: Geert Grooteplein 10, 848

department: Human Genetics

country: Netherlands

institute: UMCN St Radboud

description: Hybridized to 2.7M

hyb_protocol: DNA was restriction digested, PCR amplified, fragmented, labeled and hybridized to each array according to the manufacturer's instructions.

scan_protocol: The Arrays were then washed using Affymetrix fluidics stations, and scanned using the Gene Chip Scanner 3000.

data_processing:
The array image was acquired using Affymetrix GeneChip® Operating Software (GCOS version 1.4). Copy number values for individual SNPs were extracted and converted from CEL files into signal intensities using GTYPE 4.0 and Copy Number Analyzer for Affymetrix GeneChip mapping (CNAGT 2.0) software. The inferred copy number states were derived from a Hidden Markov Model (HMM) based algorithm implemented in CNAG2.0. Regions of homozygosity (LCSH/LOH) were determined with a likelihood based algorithm implemented in CNAG2.0.
Genotype Call (SNP call): AA, AB, BB, NC, and NoCall; 'Signal' = Summarized signal

label: Biotin

source_name: peripheral (EDTA) blood

label_protocol: As per manufacturer (Affymetrix)

growth_protocol: No culture but direct isolation from peripheral blood

extract_protocol: Genomic DNA was isloated with a Chemagen/Hamilton magnetic bead DNA isolation robot

value: female


title: 193a

value: Postmenopausal

value: Invasive ductal carcinoma

value: LumB

value: Universal Human Reference RNA (Stratagene)


title: 184a


title: 179a

value: LumA


title: 201a


title: 183a

value: BRCA2 c.6486_6489delACAA, Exon11, p.(Lys2162Asnfs*5)


title: 169a