title: ES Differentiation HD day 17_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74152.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 17 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1520, 7000; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.89; Parameter Scan Time = 00:28:56; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-18; Parameter Pixel Size = 10; Parameter Laser on time = 25298; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.26; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.89; Parameter Scan Time = 00:28:56; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-18; Parameter Pixel Size = 10; Parameter Laser on time = 24019; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.11; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 5_rep2

status: Public on Sep 09 2010

type: RNA

city: Stanford

first: Nesanet

last: Mitiku

state: CA

address: 300 Pasteur Drive Alway M313

department: Genetics

country: USA

institute: Stanford University

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74143.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 5 d

hyb_protocol: see channel 2 labeling protocol

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6840; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.79; Parameter Scan Time = 22:27:26; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-17; Parameter Pixel Size = 10; Parameter Laser on time = 25177; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.43; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.79; Parameter Scan Time = 22:27:26; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-17; Parameter Pixel Size = 10; Parameter Laser on time = 23898; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.01; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku

data_processing: VALUE is Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)

molecule: total RNA

organism: Mus musculus

source_name: REF

label_protocol: see channel 2 labeling protocol

extract_protocol: Reference RNA extraction: The common reference used in this work was comprised of a mixture of maternal brain, E17.5 embryo, E17.5 placenta, and CGR8 ES cells.  All tissues used in the reference RNA pool, with the exception of CGR8 ES cells, were ground via mortar and pestle in the presence of liquid nitrogen.  The resulting powder was put through one acid phenol extraction, two Trizol (Invitrogen) extractions, DNAse I (Ambion) treated, and then re-purified over RNAqueous Midi columns (Ambion).  The ES cell component of the reference pool was purified similarly beginning with the acid phenol extraction.  In lieu of mortar and pestle grinding, ES cells were spun down and directly resuspended in solution D.  Total RNA from all four sources was combined at a proportion of 1:1.5:2:2.5 (brain:placenta:ES cells:E17.5 embryos, according to mass) and linearly amplified (mMessageAmp, Ambion).  All reference amplification reactions were then pooled. This reference aRNA pool was used for all hybridizations.; Protocol Type = Extract preparation; Performer: Nesanet,,Mitiku

tag: REF

source_name: EXP

label_protocol:
Linear Amplification; RNA  Linear Amplification  As sample quantity and, as a result, RNA quantity was limiting , purified total RNA samples were linearly amplified (Eberwine 1996).  1000ng of total RNA were used in each amplification reaction using the mMessageAmp kit (Ambion).  First strand synthesis was carried out for 2 hours at 42degC with a heated lid (47degC) in an Eppendorf thermocycler.  Immediately after first strand synthesis, a denaturing step was carried out for 10 minutes at 70degC with the thermocycler lid heated to 80degC.  Samples were then quickly spun down and placed on ice while preparations were made for the second strand synthesis reaction.  Second strand synthesis was carried out for 2 hours at 16degC without a heated lid.  cDNA template purification was carried out per the manufacturer?s recommendations.  Synthesis of amplified RNA (aRNA) from the resulting purified cDNA template mix was carried out at 37degC for 10 hours.   aRNA samples were then treated with DNAse for 45 minutes and further purified according to manufacturer instructions.  5 mcL of the final aRNA product was examined for both sample integrity and assessment of amplification efficiency via agarose gel electrophoresis.; Protocol Type = Amplification; Performer: Nesanet,,Mitiku
Sample Labeling; Microarray Methods: Labeling and Hybridization Each labeling reaction was carried out using 4 mcg of aRNA , 10 mcg of pDN6 (Amersham), 0.6 mcL 50X dNTPs (Invitrogen), 3 mcL 0.1M DTT, 3 mcL Cy3 or Cy5 dUTP and 2mcL Superscript II (Invitrogen) reverse transcriptase in the supplied buffer.  Samples were incubated at 42°C for 2 hours in an Eppendorf cycler with the lid heated to 47°C.  Each labeling reaction was stopped via the addition of 15 mcL of 0.1N NaOH + 2mM EDTA followed by incubation at 70°C for 10 min.  15 mcL of 0.1N HCL was added to neutralize each sample.  Cy3 and Cy5 samples for a single hybridization were combined.  Labeled aRNA samples were then purified over Microcon YM-30 columns (Millipore); samples were washed 5X with TE buffer and suspended in a volume of 10.14 mcl TE by the end of purification.; Protocol Type = Labeling; Performer: Nesanet,,Mitiku

extract_protocol:
ES Cell Differentiation with LIF --> Culture conditions; Passage 13 CGR8 ES cells were grown in GMEM-based cell media (500 mL GMEM, 5 mL 200mM Glutamine, 5 mL 100mM MEM Sodium Pyruvate, 5 mL 100mM MEM Non Essential Amino Acids, 50 mL Characterized FBS (Hyclone), 50 mcL Leukemia Inhibitory factor (ESGro, Chemicon), 0.56 mL 1000X Beta-mercaptoethanol stock) in an incubator with 6% carbon dioxide at 37°C.  Sub-confluent monolayer cultures were split 1:10 every other day using trypsin.  Briefly, used growth media was removed, cells were washed once in phosphate buffered saline (Gibco BRL), trypsin solution was applied at a volume appropriate to the current flask size (25cm2 flask = 1mL; 75cm2 flask = 3mL; 150cm2 flask = 5mL), and cells were incubated in trypsin solution for 2 minutes at 37°C followed by further dissociation via pipetting.  Trypsin activity was terminated by the addition of 9 volumes of ES cell media.  The cells were then spun down at 1200g for 3 minutes swing bucket table top, diluted in fresh ES cell media, and plated on an appropriate gelatin-coated surface.  On intervening days, old growth media was exchanged for fresh pre-warmed ES cell media. In preparation for differentiation, sub-confluent ES cells were split 1:15 two days prior to the start of the differentiation timecourse.  This resulted in 70% confluence on day 0 of the differentiation timecourse.  Cells were released from the flask surface by trypsinization as described above.  Differentiation media was identical to the standard ES cell media.  Cell counts were assessed twice in preparation for hanging drop cultures: once of the stock resulting from trypsin inactivation and once of a 1:10 dilution of that stock.  The average of the two calculations was used as the final post-trypsinization cell concentration.  The ES cells were then diluted in media in order to achieve a concentration approximately 880 cells per 25 mcL volume.  Forty drops of 25 mcL cell suspension were applied to the lid of each 10cm Petri dish.  Each lid was then carefully inverted and placed over its corresponding Petri dish bottom.  The cell suspension drops were thus ?hanging drops.?  Each Petri dish bottom contained 10mL of prewarmed (37°C) PBS in order to keep the drops adequately hydrated.  Within each drop, resident cells move toward the lowest point and aggregate, forming embryoid bodies (EBs) within the first day of suspension culture.   After two days in hanging drop culture, EBs were transferred to ordinary suspension culture.  For this purpose, the appropriate number of 6 well Costar low adhesion plates (Corning) were pre-incubated with fresh prewarmed differentiation media for greater than 20 minutes at 37°C.  Subsequently, 20 EBs were transferred to each well of a 6-well plate.; Protocol Type = Growth Conditions; Performer: Nesanet,,Mitiku
Sample Extraction: Experimental Sample RNA Isolation  Total RNA was prepared using a variation on the acid phenol method of Chomczynski et al(Chomczynski and N. 1987).  The method of sample homogenization in solution D (4M guanidinium thiocyanate, 100mM NaOAc, 0.5% Sarkosyl, 0.1M beta-mercaptoethanol)  varied according to the composition of the sample from which RNA was being extracted.  Suspension growth embryoid bodies (EBs) were transferred into microfuge tubes, washed twice with phosphate buffered saline, and then resuspended in solution D; homogenization was achieved through pipetting.  Adherent cells were washed twice with phosphate buffered saline, lysed on the plate with solution D, scraped vigorously with a cell scraper, passed 2-3times through a 27 gauge needle, and then transferred to a microfuge tube.  Homogenized samples were stored at -80degC until RNA isolation.   On the day of RNA isolation, solution D-homogenized samples were defrosted and vortexed to ensure complete mixing.  As the initial volume of lysate was in accordance to the initial sample quantity, lysate volumes were variable, and solution volumes throughout the RNA isolation were adjusted for the initial lysate volume.  Initial lysate volumes were one of the following: 650 mcL/400 mcL/300 mcL/100 mcL.  Lysates were stored at -80degC.  Phase Lock Gel-Heavy (PLGH) tubes were prespun at 1500g for 30sec in order to collect the gel at the bottom of the microfuge tube.  65 mcL/40 mcL/30 mcL/10 mcL NaOAc pH 4.0 was added to each defrosted sample, followed by 400mcL H20-saturated phenol.  The lysate-NaOAc-phenol mix was added to the PLGH tubes and incubated for 5 minutes at room temperature.  487.5 mcL/300 mcL/225 mcL/75 mcL chloroform:isoamyl alcohol was added to each differentiation timecourse sample.  Each tube was shaken vigorously for 15 seconds and then less vigorously for 2 minutes.  Samples were incubated for 15 minutes at room temperature and the centrifuged at 12000g for 15 minutes at room temperature.  The clear aqueous phase above the Phase Lock Gel layer was carefully removed and transferred into a fresh nuclease-free tube.  In order to precipitate the RNA, the following were added to each sample: 1/10th vol 5mcL linear acrylamide (Ambion), and 1X vol isopropanol.  Samples were then incubated on dry ice or -80degC for a minimum of 1 hour.  After defrosting, 1/10th vol NaOAc pH 4.0 was added to each sample, followed by 1X vol mcL H20-saturated phenol.  1/5th vol of chloroform:isoamyl alcohol was added to each sample.  Samples were mixed via inversion then incubated on ice for 15 minutes.  After this incubation, samples were centrifuged at 20817g for 15 minutes at room temperature.  The top phase of each sample was removed and deposited in a fresh nuclease-free tube.  Samples were then precipitated at -80degC using 1/10th vol NaOAc, 5 mcL linear acrylamide (Ambion), and 1X vol isopropanol.   Prior to starting the second round of purification, the samples were defrosted on ice, and centrifuged at 20817g for 30 minutes at room temperature.  After removal o f all isopropanol, the resulting pellet was resuspended in sample lysis buffer (RNAqueous Mini kit, Ambion).  During this step, the provided silica-based filter cartridges were assembled in microfuge tubes.  Following dissolution of the pellet, 400mcL of 64% ethanol was added.  Mixing was achieved through pipeting 2-3 times.  The resulting mixture was immediately applied to the previously prepared filter cartridges.  After centrifugation at 13000g for 1 minute, the flow-through was discarded.  Subsequently, each cartridge was washed once with 700mcL of Wash Solution #1 and twice with 500 mcL of Wash Solution #2.  Cartridges were spun for 1 minute in the absence of wash solution in order to remove any residual ethanol.  Purified RNA was eluted into a fresh nuclease free tube with nuclease-free H20 preheated to 75degC.; Protocol Type = Extract preparation; Performer: Nesanet,,Mitiku

tag: EXP


title: ES Differentiation HD day 1_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74153.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 1 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1560, 6600; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.89; Parameter Scan Time = 22:17:29; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-08; Parameter Pixel Size = 10; Parameter Laser on time = 26621; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.73; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.89; Parameter Scan Time = 22:17:29; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-08; Parameter Pixel Size = 10; Parameter Laser on time = 25342; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.10; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 9_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74162.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 9 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1560, 7040; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.36; Parameter Scan Time = 00:52:58; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 26776; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 1.99; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.36; Parameter Scan Time = 00:52:58; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 25497; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.15; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 5_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74128.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 5 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1400, 6640; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.64; Parameter Scan Time = 23:06:34; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 25580; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.19; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.64; Parameter Scan Time = 23:06:34; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 24302; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.02; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 0_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74137.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 0 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6720; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 35.39; Parameter Scan Time = 21:40:39; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 26028; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.12; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 35.39; Parameter Scan Time = 21:40:39; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 24749; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.00; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 4_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74127.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 4 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6480; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 33.86; Parameter Scan Time = 20:39:15; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 25964; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.76; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 33.86; Parameter Scan Time = 20:39:15; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 24686; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.94; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 6_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74129.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 6 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1400, 6320; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.56; Parameter Scan Time = 00:04:44; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25642; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.68; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.56; Parameter Scan Time = 00:04:44; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24363; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.10; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 7_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74145.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 7 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1560, 6880; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.19; Parameter Scan Time = 23:03:22; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-17; Parameter Pixel Size = 10; Parameter Laser on time = 25213; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.32; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.19; Parameter Scan Time = 23:03:22; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-17; Parameter Pixel Size = 10; Parameter Laser on time = 23934; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.07; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 4_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74142.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 4 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1520, 6760; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.86; Parameter Scan Time = 22:56:28; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 26103; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.43; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.86; Parameter Scan Time = 22:56:28; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 24824; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.07; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 13_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74134.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 13 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6720; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.96; Parameter Scan Time = 01:22:46; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25719; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.16; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.96; Parameter Scan Time = 01:22:46; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24440; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.16; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 15_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74135.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 15 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6640; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.14; Parameter Scan Time = 01:37:33; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25733; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.01; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.14; Parameter Scan Time = 01:37:33; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24455; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.17; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 11_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74164.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 11 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1640, 6520; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.45; Parameter Scan Time = 01:06:53; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 26789; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 1.85; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.45; Parameter Scan Time = 01:06:53; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 25511; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.16; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 8_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74146.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 8 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1640, 7000; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.31; Parameter Scan Time = 23:16:00; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-17; Parameter Pixel Size = 10; Parameter Laser on time = 25225; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.26; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.31; Parameter Scan Time = 23:16:00; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-17; Parameter Pixel Size = 10; Parameter Laser on time = 23946; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.08; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 3_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74141.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 3 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1440, 6400; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.67; Parameter Scan Time = 22:43:44; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 26090; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.53; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.67; Parameter Scan Time = 22:43:44; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 24812; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.06; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 15_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74150.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 15 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1360, 6360; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.83; Parameter Scan Time = 00:13:25; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-18; Parameter Pixel Size = 10; Parameter Laser on time = 25283; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.40; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.83; Parameter Scan Time = 00:13:25; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-18; Parameter Pixel Size = 10; Parameter Laser on time = 24004; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.11; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 2_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74125.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 2 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1400, 6280; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 35.63; Parameter Scan Time = 22:23:53; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 25540; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.24; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 35.63; Parameter Scan Time = 22:23:53; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 24261; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.99; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: CDM_AM53_3

status: Public on Sep 08 2010

city: Columbus

first: Chuck

last: Daniels

state: OH

address: 484 West 12th Avenue

department: Microbiology

institute: The Ohio State Uinversity

description: specialized growth conditions for this sample: none

hyb_protocol: Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 50° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 100μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 50° at 60rpm• Fluidics washing is FS450_0001

scan_protocol: Affymetrix Gene ChIP Scanner 3000 7G

data_processing: CEL files were generated from scanned images (DAT files) using GCOS software (Affymetrix). Probe set signals were generated using an in-house developed WPP algorithm. WPP (Well behaved estimates of differential gene expression Plus probe-level p-values Plus extensible quantile scaling; Auer, H. et. al. 2007) software is an enhanced version of RMA (Irizarry R.A., Bolstad B.M., Collin F., Cope L.M., Hobbs B., Speed T.P. Nucleic Acids Res. 2003, 31(4):e15). Data are presented as quantile scaled probe level expression values (Auer, H. et. al. 2007) for PM-MM. If PM-MM was ≤0, the value was set at 1.0.  Pooled samples are presented as quantiled normailized probe level expression values (Bolstad, B.M. et. al. 2003) for PM-MM. If PM-MM was ≤0, the value was set at 1.0.

label: Biotin

organism: Haloferax volcanii

source_name: H53, Completely defined media, succinate/glycerol, exponential

label_protocol: 4.0ug of total RNA is randomly primed at 75 degrees for 10 minutes (3ug/ul Random Primers, Invitrogen, Carlsbad, USA).  SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, USA) is used for first strand cDNA synthesis following manufacturer’s instructions. The first strand cDNA is purified and content is measured in a ND-1000 spectrophotometer.  ~6ug of the purified product is then fragmented/labeled according to Affymetrix Genechip Mapping 100K assay manual (Affymetrix Inc., USA).

growth_protocol: H. volcanii DS70 (Wendoloski et al. 2001) was cultured aerobically in baffled flasks and foam stoppers in completely defined (CDM) or complex (CX) media at 42°C with constant shaking at 220 rpm. CDM contained the following components (per liter): 125 g NaCl, 50 g MgCl2•6H20,  5 g K2SO4, 0.26 g CaCl2•2H20. After autoclaving, the following components were added to cooled media to the indicated final concentration: 16.7 mM Na-succinate, 4.3 mM glycerol, 10 mM NH4Cl, 2 ml of 0.5 M KHPO4, 1ml Balch trace elements (Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS. Microbiol Rev. 1979, 43: 260-96.), 100 μl of 0.1 mg/ml biotin stock, 100 μl of 0.8 mg/ml thiamine stock.  H. volcanii strains ASD40, ASD41 and H53 grown in CDM were supplemented with uracil (50 μg/ml).  CX contained the following components (per liter): 125 g NaCl, 45 g MgCl2•6H20, 10 g MgSO4•7H20, 10 g KCl, 1.34 g CaCl2•2H20, 3 g yeast extract and 5 g tryptone.  For balanced growth experiments (exponential) the cultures were grown to an OD550 of 0.1.

extract_protocol: Total cellular RNA was isolated from H. volcanii strains using a modified protocol of the TRIzol® reagent. Aliquots of 50 ml culture samples were placed into 50 ml Falcon tubes and the cells were harvested by centrifugation at 5500 rpm for 5 minutes at 4°C in the Beckman JA20.25 rotor. Pellets were resuspended in 500 μl spheroplasting solution (Cline, S.W., Schalkwyk, L.C., Doolittle, W.F. PNAS. 1989, 171: 4987-4991) and transferred to a 1.5 ml microcentrifuge tube. Total RNA was extracted by first adding 500 μl TRIzol® reagent and incubating at room temperature for 5 minutes. Then 200 μl of chloroform was added, the mixture was mixed on a vortex mixer for 30 seconds and incubated at room temperature for 3 minutes. The two phases were separated by centrifugation at 14,000 rpm for 10 minutes at 4°C in a microcentrifuge. The top aqueous phase was collected and placed in a microcentrifuge tube, one volume of ice-cold isopropanol was added and the mixture was incubated at room temperature for 10 minutes. The precipitated RNA was pelleted by centrifugation at 14,000 rpm for 10 minutes at 4°C in a microcentrifuge. The RNA pellet was allowed to dry and was resuspended in 200 μl ddH2O.  The TRIzol® reagent extraction procedure was repeated to remove residual DNA and protein and the final sample was resuspended in 200 μl ddH2O.      An extended protocol was used for the preparation of small RNAs in the experiments CDM_sRNAP_E, CDM_sRNAP_S, CX_sRNAP_E and CX_sRNAP_S. Three independent H. volcanii DS70 100 ml cultures were grown for each of the four conditions (CDM_sRNAP_E, CDM_sRNAP_S, CX_sRNAP_E, CX_sRNAP_S).  At the appropriate point in growth curve (A550 of 0.1 for exponential CDM and CX, and OD550 of 1.0 or 4.8, for stationary CDM or CX, respectively) the three related cultures were then combined into a single 300 ml culture. Small RNA enrichments were prepared from the individual pooled samples.  Total RNA was prepared using the modified TRIzol® reagent method and RNAs were further processed using a modification of the method described for the QIAGEN-tip 100 column.  QIAGEN-tip 100 columns were equilibrated with 15 ml of equilibration buffer (0.05M MOPS pH 7, 15% isopropanol, 1% Triton). RNA samples were brought to 0.1M MOPS pH 7, then passed over the column four times to facilitate binding of small RNAs. The column was washed two times with 15 ml wash buffer (0.05M MOPS pH 7, 0.2M NaCl) and small RNAs were eluted in 1.5 ml fractions by running 15 ml of elution buffer (0.05M MOPS pH 7, 0.75M NaCl, 15% ethanol) over the column.  Fractions two and three contained the bulk of RNA eluted from the column. RNA was recovered by precipitation with and equal volume of isopropanol and resuspended in 200 μl ddH20. These RNA preparations were enriched in RNAs below 500 nt.  For DNA hybridization total genomic DNA was prepared from two independent 10 ml H. volcanii DS70 cultures grown in CDM to a final density of A550 of 1.0. Cells were harvested by centrifugation at 5500 rpm for 10 minutes at 4°C in the Beckman JA20.25 rotor in 50 ml Falcon tubes. To lyse the cells, the pellet was resuspended in 10 ml of ddH2O with 1% SDS and the nucleic acids were precipitate with the addition of two volumes of isopropanol.  Precipitated nucleic acids were spooled onto a glass rod and placed into a 1.5 ml centrifuge tube and pelleted by centrifugation at 14000 rpm for 10 minutes at 4°C. The pellet was resuspended in 200 μl of ddH2O. RNA was removed by adding 3 μl of a 20 μg/μl DNase free RNase A solution (Invitrogen) followed by incubated at 37°C for 30 minutes. The RNase was removed by phenol:chloroform extraction and the DNA was recovered by ethanol precipitation. The precipitated DNA was resuspended in 200 μl of ddH2O.

tag: strain


title: ES Differentiation HD day 11_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74133.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 11 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1400, 6360; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.66; Parameter Scan Time = 01:06:23; Parameter PMT Volts = 550; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25703; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.38; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.66; Parameter Scan Time = 01:06:23; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24424; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.13; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: DNA1

type: genomic

molecule: genomic DNA

source_name: DS70, DNA, Completely defined media, succinate/glycerol, stationary


title: ES Differentiation HD day 17_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74167.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 17 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6120; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.60; Parameter Scan Time = 01:50:12; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 26833; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 1.48; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.60; Parameter Scan Time = 01:50:12; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 25554; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.16; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 2_rep2

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74140.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 2 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1600, 6360; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.37; Parameter Scan Time = 22:26:16; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 26073; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.35; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 36.37; Parameter Scan Time = 22:26:16; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-01; Parameter Pixel Size = 10; Parameter Laser on time = 24794; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.03; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: DNA2

source_name: DS70, DNA, completely defined media, succinate/glycerol, stationary


title: CDM_AM53_2


title: ES Differentiation HD day 9_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74132.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 9 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1320, 6360; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.41; Parameter Scan Time = 00:51:22; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25687; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.90; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.41; Parameter Scan Time = 00:51:22; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24409; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.13; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 0_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74123.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 0 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6440; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 34.26; Parameter Scan Time = 21:38:01; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 25494; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.33; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 34.26; Parameter Scan Time = 21:38:01; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 24216; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.96; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ASD41_H

description: specialized growth conditions for this sample: 5 mM histidine replaced ammonium ion as sole nitrogen source

source_name: ASD41, Completely defined media, succinate/glycerol, exponential, histidine as sole nitrogen source (pooled)


title: CDM_AM1


title: CDM_sRNAP_S

source_name: DS70, small RNA, completely defined media, succinate/glycerol, stationary (pooled)


title: ES Differentiation HD day 5_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74158.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 5 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1560, 6720; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.87; Parameter Scan Time = 23:48:39; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-08; Parameter Pixel Size = 10; Parameter Laser on time = 26711; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 1.84; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.87; Parameter Scan Time = 23:48:39; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-08; Parameter Pixel Size = 10; Parameter Laser on time = 25433; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.12; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 6_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74159.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 6 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1560, 7240; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.08; Parameter Scan Time = 00:08:11; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 26729; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 1.76; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.08; Parameter Scan Time = 00:08:11; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 25451; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.13; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: CX_sRNAP_E

source_name: DS70, small RNA, complex media, exponential (pooled)


title: ES Differentiation HD day 7_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74130.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 7 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1400, 6360; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.89; Parameter Scan Time = 00:23:11; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25659; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.15; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 37.89; Parameter Scan Time = 00:23:11; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24381; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.12; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: CX_sRNAP_S

source_name: DS70, small RNA, complex media, stationary (pooled)


title: ASD40_H

source_name: ASD40, Completely defined media, succinate/glycerol, exponential, histidine as sole nitrogen source (pooled)


title: CDM_AM3


title: ASD40

source_name: ASD40, Completely defined media, succinate/glycerol, exponential (pooled)


title: CDM_AM53_1


title: ES Differentiation HD day 8_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74131.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 8 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6640; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.14; Parameter Scan Time = 00:37:31; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 25674; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.62; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.14; Parameter Scan Time = 00:37:31; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-25; Parameter Pixel Size = 10; Parameter Laser on time = 24395; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.12; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: ES Differentiation HD day 1_rep1

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74124.gif
Measurement: concentration(absolute) 0 volume/volume_percent
Measurement: time(absolute) 1 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1400, 6240; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 34.69; Parameter Scan Time = 21:51:54; Parameter PMT Volts = 540; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 25508; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.38; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 34.69; Parameter Scan Time = 21:51:54; Parameter PMT Volts = 660; Parameter Scan Date = 2005-04-24; Parameter Pixel Size = 10; Parameter Laser on time = 24229; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.96; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: CDM_AM2


title: ASD41

source_name: ASD41, Completely defined media, succinate/glycerol, exponential (pooled)


title: CDM_sRNAP_E

source_name: DS70, small RNA, completely defined media, succinate/glycerol, exponential (pooled)


title: CDM_CR3

description: specialized growth conditions for this sample: A starter culture of H. volcanii DS70 was used as the inoculum (1:1000 dilution) in eight 300 ml cultures of CDM in baffled flasks with foam stoppers (42‹C; 220 rpm). The cultures were grown to an OD550 of 0.1 and the cells were pelleted in sterile centrifuge tubes by centrifugation at 5,000 rpm for 10 minutes at 4‹C in the Beckham JLA10.500 rotor. The cell pellets were washed with basal salts and pelleted by centrifugation as stated above. The supernatant was removed and four of the cell pellets were resuspended in 300 ml of CDM supplemented with succinate and glycerol as carbon sources (CDM_CR). The other four cell pellets were individually resuspended in 300 ml of CDM; however, no carbon source was added (CDM_C). Growth was monitored by the turbidity at 550 nm. Once the four CDM_CR had doubled in turbidity, the culture was allowed to grow for an additional hour. The CDM_C cultures were incubated an equivalent period of time as the CDM_CR cultures. This corresponded to an incubation period of 3.5 hours.  RNA was prepared from each and three biological replicates (CDM_C and CDM_CR) were used in array analysis.

source_name: DS70, Completely defined media, succinate/glycerol, exponential, control recovery for carbon starvation


title: CDM_CR1


title: CDM_CR2


title: ES Differentiation HD day 15_withLIF

description:
Simple annotation: Development, development
Image: http://smd.stanford.edu/MicroArray/gifs/2007-05/74166.gif
Measurement: concentration(absolute) 0.1 volume/volume_percent
Measurement: time(absolute) 15 d

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1480, 6240; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Nesanet,,Mitiku Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.54; Parameter Scan Time = 01:36:23; Parameter PMT Volts = 540; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 26820; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 2.04; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 39.54; Parameter Scan Time = 01:36:23; Parameter PMT Volts = 660; Parameter Scan Date = 2005-05-09; Parameter Pixel Size = 10; Parameter Laser on time = 25541; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [82695]; Parameter Laser Power = 3.16; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Nesanet,,Mitiku


title: CDM_NR3

description: specialized growth conditions for this sample: A starter culture of H. volcanii DS70 was used as the inoculum (1:1000 dilution) in eight 300 ml cultures of CDM in baffled flasks with foam stoppers (42‹C; 220 rpm). The cultures were grown to an OD550 of 0.1 and the cells were pelleted in sterile centrifuge tubes by centrifugation at 5,000 rpm for 10 minutes at 4‹C in the Beckham JLA10.500 rotor. The cell pellets were washed with basal salts and pelleted by centrifugation as stated above. The supernatant was removed and four of the cell pellets were resuspended in 300 ml of CDM supplemented with succinate and glycerol as carbon sources and 10 mM ammonium chloride (CDM-NR). The other four cell pellets were individually resuspended in 300 ml of CDM supplemented with succinate and glycerol as carbon sources however, no nitrogen source was added (CDM-N). Growth was monitored by the turbidity at 550 nm. Once the four CDM-NR had doubled in turbidity, the culture was allowed to grow for an additional hour. The CDM-N cultures were incubated an equivalent period of time as the CDM-NR cultures. This corresponded to an incubation period of 3.5 hours. RNA was prepared from each and three biological replicates (CDM_N and CDM_NR) were used in array analysis.

source_name: DS70, Completely defined media, succinate/glycerol, exponential, control recovery for nitrogen starvation


title: CDM_C1

source_name: DS70, Completely defined media, succinate/glycerol, exponential, carbon starvation


title: CDM_C3


title: CDM_NR2


title: CDM_N2

source_name: DS70, Completely defined media, succinate/glycerol, exponential, nitrogen starvation


title: CDM_N3


title: CDMH1

description: specialized growth conditions for this sample: 5 mM histidine replaced 10 mM NH4Cl as sole nitrogen source

source_name: DS70, Completely defined media, succinate/glycerol, exponential, histidine as sole nitrogen source


title: CDMH2


title: CDM_N1


title: CDM_C2


title: CXS1

description: specialized growth conditions for this sample: Cells were grown to an OD550 of 4.8.

source_name: DS70, Complex media, stationary


title: CDMH3


title: CDMT2

description: specialized growth conditions for this sample: Cells were grown to an OD550 of 0.85.

source_name: DS70, Completely defined media, succinate/glycerol, transition to stationary


title: CDMS1

description: specialized growth conditions for this sample: Cells were grown to an OD550 of 1.1.

source_name: DS70, Completely defined media, succinate/glycerol, stationary


title: CXS3


title: CXS2


title: CDM25_1

description: specialized growth conditions for this sample: Final NaCl concentration in CDM was 25 g/L or 4.3 M.

source_name: DS70, Completely defined media, succinate/glycerol, 25% salt, exponential


title: CDM20_3

description: specialized growth conditions for this sample: Final NaCl concentration in CDM was 20 g/L or 3.4 M.

source_name: DS70, Completely defined media, succinate/glycerol, 20% salt, exponential


title: CDM25_2


title: CDM25_3


title: CDMS3


title: CDM20_2


title: CDM10_3

description: specialized growth conditions for this sample: Final NaCl concentration in CDM was 10 g/L or 1.8 M.

source_name: DS70, Completely defined media, succinate/glycerol, 10% salt, exponential


title: CDMT1


title: CDMT3


title: CDM20_1


title: CDMS2


title: CDML3

description: specialized growth conditions for this sample: 25 mM lactate replaced Na-succinate and glycerol as sole carbon source

source_name: DS70, Completely defined media, lactate, exponential


title: CDM10_2


title: CDM_NR1


title: CDM10_1


title: CDMP2

description: specialized growth conditions for this sample: 25 mM pyruvate replaced Na-succinate and glycerol as sole carbon source

source_name: DS70, Completely defined media, pyruvate, exponential


title: CDML2


title: CDMP3


title: Bid_KO_pMG_LPS_3H

status: Public on Sep 01 2010

city: Tel Aviv

first: Reuven

last: Stein

address: George S. Wise Faculty of Life Sciences,

country: Israel

institute: Tel - Aviv University

description:
All experiments were performed using Affymetrix  Mouse Gene 1.0st oligonucleotide arrays. as described at (
http://www.affymetrix.com/support/technical/datasheets/gene_1_0_st_datasheet.pdf

hyb_protocol: The target cDNA generated from each sample was processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf). Briefly, spike controls were added to 5.5 µg fragmented cDNA before overnight hybridisation. Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner. A complete description of these procedures is available at (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf).

scan_protocol: Array was scanned on an Affymetrix GeneChip scanner. A complete description of these procedures is available at (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf

data_processing:
Gene level RMA sketch algoritm (Affymetrix Expression Console and Partek Genomics Suite 6.2 .) can be used for crude data generation.
Genes are analysed using unsupervised hierachical cluster analysis(Spotfire DecisionSite for Functional Genomics; Somerville,MA) to get a first assessment of the data, and filtered according to fold change calculations.

label: biotin

source_name: Microglia cells

label_protocol:
Total RNA from each sample was used to prepare biotinylated target DNA, according to manufacturers recommendations (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf
Briefly, 100-600 ng of Total RNA was used to generate first-strand cDNA by using a T7-random hexamers primer. After second-strand synthesis, in vitro transcription was performed. The resulting cRNA is then used for  a second cycle  of first-strand cDNA by using a T7-random hexamers primer with UTP resulting in SS DNA used for fragmentation and terminal labeling

extract_protocol:
total RNA was extracted using Trizol reagent (Invitrogene) according to the manufacture instructions, folowed by Total RNA purification using phenol-chlorophorm extraction and Phase Lock Gel as deatiled in the this protocol.
1.	Equilibrate the phenol:chlorophorm:isoamyl alcohol mix to room temperature (may take 10-15 min.)
2.	2. Pellet the Phase lock gel (PLG) tubes prior to use by centrifugation 1 minute at maximum speed.
3.	To the RNA dissolved in 400 ul water, add an equal volume (400 ul) of 25:24:1 phenol:chlorophorm:isoamyl alcohol mix(saturated with 10mM Tris-HCl pH 8.0/1mM EDTA, GIBCO) , and vortex 4-5 seconds.
4.	Transfer the entire Total RNA –phenol/chlorophorm mixture to the PLG tube (DO NOT VORTEX. PLG will not become part of the suspension. Microcentrifuge at full speed for 2 minutes.
5.	Transfer the aqueous upper phase to a fresh 1.5 ml tube. Pay attention not to touch the gel with the pipet tip as it lowers grade of RNA purity. Hold PLG tubes securely when transferring -they are extremely slippery!
6.	Repeat steps 3, 4 and 5.
7.	Precipitate the RNA by adding 0.1 vol 3M sodium acetate (RNAse free, Sigma) and 0.8 volume isopropanol (RNA vol.+sodium acetate vol.)  For 400 ul RNA: 40 ul Sodium acetate and 352 ul isopropanol, and vortex.
8.	Store at –20C overnight.
9. Fast cool the centrifuge. Spin the samples at full speed for 20 min at 4 C. Always place the tubes with a cap tip toward the outermost side of the rotor, then the position of  RNA pellet would be at the bottom on this outermost side. Pipet off the supernatant, take care not to lose he pellet.
9.	Rinse the pellet with 0.5 ml 80% ethanol (made with DEPC-treated water , stored at –20C), 5 min. 9500 rpm 4C.  Pipet out the supernatant. Repeat wash! (5 min.)
10.	Pipet out the ethanol using tips in descending volume order: tip of 1000 ul, then 200 (100) ul, then short spin the samples in Eppendorf centrifuge at  full speed 10 sec., and pipet out the remainder of ethanol by the finest tip (10 Reach) – take care not to touch the pellet!
On this step, pay attention to the pellet size – it helps to estimate DDW volume  for resuspension. Very small pellets are to be resuspended in 13 ul of water. Larger quantities of RNA may be resuspended in 20-30 ul of water.
Air dry the pellets (10 min. room temperature, open tubes carefully and let them stay in a clean secure place )
 Simultaneously, preheat the DEPC treated water to 70C.
Check the RNA for dryness. When dry, it is almost  fully transparent. Dry samples extra time if needed.  Add apprpopriate volume of DDW to the pellet – do not mix! Heat  the samples 10 min. at 70 C. Then mix well by pipetation.

tag: genotype

value: Bid-/-, KO

tag: agent

tag: cell type

value: microglial cells


title: CDM3

description: specialized growth conditions for this sample: none

source_name: DS70, Completely defined media, succinate/glycerol, exponential


title: Human_PBMNCs_KD_patients_669

status: Public on Sep 04 2010

city: Fukuoka

first: Kazuyuki

last: Ikeda

state: Fukuoka

address: Maidashi 3-1-1

department: Pediatrics

country: Japan

institute: Kyushu university

description: Blood samples were obtained prior to the treatment from patients with Kawasaki disease, and from 5 healthy adults. PBMNCs were separated from peripheral blood.

hyb_protocol: Samples were applied to microarrays, and slides were hybridized for 20 h at 42C.

scan_protocol: The arrays were scanned by FLA-8000 (Fuji Photo Film,Tokyo, Japan) Images were quantified using ArrayVision (Amersham Biosciences).

data_processing: The numerical data were normalized using LOWESS method. The data with low signal-to-noise ratios (S/N<3) were not used for further analysis, so the sample data tables contain only 22839/30336 features of the array. The PRE_VALUE data were used in the study.

organism: Homo sapiens

source_name: human PBMNCs from patients with Kawasaki disease

label_protocol: Double-strand cDNA was synthesized from total RNA using oligo dT primer with a T7 RNA polymerase promoter site added to the 3’ end. Then, in vitro transcription was performed in the presence of amino allyl UTP to produce multiple copies of amino allyl labelled  cRNA. Amino allyl labelled cRNA was purified, and then reacted with N-hydroxy succinimide esters of Cy3 (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for cRNA from PBMNCs of healthy controls, and Cy5 (Amersham Pharmacia Biotech) for that from PBMNCs of the acute-phase KD patients.

extract_protocol: Total RNA was extracted from these cells using an RNA extraction kit (Isogen; Nippon Gene, Osaka, Japan), according to the manufacturer’s instruction.

value: PBMNCs

tag: disease state

value: patients with Kawasaki disease

source_name: human PBMNCs from healthy controls

value: healthy controls


title: M15 - infected MBT/Pas fibroblasts (9 h post inf)

status: Public on Aug 31 2010

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

country: Germany

institute: HCI - Helmholtz Centre for Infection Research

hyb_protocol: Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at conditions recommended by the manufacturer. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Affymetrix GCS 3000.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 150.

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: RNAeasy

value: MBT/Pas

value: mouse embryonic fibroblasts (MEFs)

tag: rift valley fever virus

value: infected


title: CX3

source_name: DS70, Complex media, exponential


title: M17 - infected MBT/Pas fibroblasts (9 h post inf)


title: CDMP1


title: CX2


title: CX1


title: Human_PBMNCs_KD_patients_663


title: CDM2


title: Human_PBMNCs_KD_patients_662


title: pMG_C57Bl_LPS_3H

value: wildtype


title: M16 - infected MBT/Pas fibroblasts (9 h post inf)


title: pMG_C57Bl_control


title: M13 - non-infected MBT/Pas fibroblasts

description: M13 - non-infected MBT/Pas fibroblasts

source_name: non-infected MBT/Pas fibroblasts

value: non-infected


title: M12 - non-infected MBT/Pas fibroblasts

description: M12 - non-infected MBT/Pas fibroblasts


title: M11 - non-infected MBT/Pas fibroblasts

description: M11 - non-infected MBT/Pas fibroblasts


title: CDML1


title: CDM1


title: B17 - infected BALB/cByJ fibroblasts (9 h post inf)

value: BALB/cByJ


title: Bid_KO_pMG_Control


title: B13 - non-infected BALB/cByJ fibroblasts

source_name: non-infected BALB/cByJ fibroblasts


title: B12 - non-infected BALB/cByJ fibroblasts


title: B15 - infected BALB/cByJ fibroblasts (9 h post inf)


description: B11 - non-infected BALB/cByJ fibroblasts.


title: B16 - infected BALB/cByJ fibroblasts (9 h post inf)


title: mouse_dermis_rep1

status: Public on Aug 29 2010

city: New York

first: Roy

last: Blum

state: NY

address: 552 First Ave

department: Cancer Institute

institute: New York University

hyb_protocol: Ten micrograms of labeled and fragmented cRNA were then hybridized at 45°C for 16 h to the mouse genome MOE430 2.0 array (Affymetrix) which interrogates ~45,000 transcripts.

scan_protocol: Affymetrix scan protocol

data_processing: Raw expression data were analyzed using GCOS 1.4 (Affymetrix). Data were normalized to a target intensity of 500 to account for differences in global chip intensity.

source_name: Total RNA of mouse skin dermis

label_protocol: Samples with a RNA Integrity Number (RIN) >7.0 were considered suitable for labeling and 20 ng were labeled using the GeneChip two-cycle target labeling kit (Affymetrix).

growth_protocol: 16-day-old C57BL/6 murine embryos were used.

extract_protocol: RNA was isolated by chloroform extraction and precipitated using isopropanol. Additional column purification was conducted (RNeasy mini kit, Qiagen) and the resulting RNA treated to remove DNA contamination (DNAfree, Ambion). RNA concentration was assessed spectrophotometrically using a NanoDrop® ND-1000 Spectrophotometer (Wilmington). Protocols produced clean RNA with OD 260/280 >1.7. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies).

treatment_protocol: UGE and UGM were isolated from the urogenital sinus of 16-day-old C57BL/6 murine embryos after digestion with trypsin and added to TRIzol.  Epidermis and dermis were prepared from skin removed from the back of E16 mouse embryos. Skin was incubated overnight at 4C in 2 ml of Dispase solution (Gibco, cat # 17105-041, 0.4 mg/ml) in DMEM containing 10% FCS. The dermis and epidermis were separated manually under the microscope and added to TRIzol.

tag: stage


title: mouse_epidermis_rep2

source_name: Total RNA of mouse skin epidermis


title: mouse_urogenital_sinus_mesenchyme_rep4

source_name: Total RNA of mouse urogenital sinus mesenchyme


title: HH 3

status: Public on Aug 24 2010

city: Jacksonville

first: Tushar

last: Patel

state: FL

address: 4500 San Pablo Road

institute: Mayo Clinic

description: ucRNA profiling from human hepatocytes cells

hyb_protocol: Labeled targets from 5 μg of total RNA was used for hybridization on a custom microarray (OSU-CCC 4.0), which includes sense and antisense probes to all 481 human ultraconserved reported by Bejerano et al . All probes on these microarrays are 40-mer oligonucleotides spotted by contacting technologies and covalently attached to a polymeric matrix. The microarrays were hybridized in 6× SSPE (0.9 M sodium chloride/60 mM sodium phosphate/8 mM EDTA, pH 7.4)/30% formamide at 25°C for 18 h, washed in 0.75× TNT (Tris·HCl/sodium chloride/Tween 20) at 37°C for 40 min, and processed by using direct detection of the biotin-containing transcripts by Streptavidin-Alexa647 conjugate. Processed slides were scanned by using a PerkinElmer ScanArray XL5K Scanner with the laser set to 635 nm, at power 80 and PMT 70 settings, and a scan resolution of 10 μm.

scan_protocol: Biotin-containing transcripts were detected using streptavidin–Alexa647 conjugate, scanned and analyzed using an Axon 4000B scanner and the GenePix 6.0 software (Axon Instruments, Downingtown, PA)

data_processing: The mean fluorescence intensity of replicate spots were subtracted from background and normalized using the global median method. We selected ucRNAs measured as present in all the three replicates. Differentially expressed ucRNAs were identified using the Class Comparison Analysis of BRB tools version 3.6.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html).  The criterion for inclusion of a gene in the gene list was a p-value < 0.05.

source_name: human hepatocytes

label_protocol: Five micrograms of total RNA were separately added to reaction mix in a final volume of 12 μl, containing 1 μg of [3′-(N)8-(A)12-biotin-(A)12-biotin-5′] oligonucleotide primer. The mixture was incubated for 10 min at 70°C and chilled on ice. With the mixture remaining on ice, 4 μl of 5× first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP mix, and 1 μl of SuperScript II RNaseH- reverse transcriptase (200 units/μl) was added to a final volume of 20 μl, and the mixture was incubated for 90 min in a 37°C water bath. After incubation for first-strand cDNA synthesis, 3.5 μl of 0.5 M NaOH/50 mM EDTA was added into 20 μl of first-strand reaction mix and incubated at 65°C for 15 min to denature the RNA/DNA hybrids and degrade RNA templates. Then, 5 μl of 1 M Tris·HCI (pH 7.6, Sigma) was added to neutralize the reaction mix, and labeled targets were stored in 28.5 μl at -80°C until chip hybridization.

extract_protocol: trizol protocol

treatment_protocol: cells were cultures in 100 mm dishes and collected for RNA extraction

tag: cell line

value: normal human hepatocytes


title: HepG2 3

description: ucRNA profiling from HepG2 cells

source_name: HepG2 human hepatoma cell line


title: mouse_urogenital_sinus_mesenchyme_rep1


title: HepG2 2

description: ucRNA profiling from HepG2 cells


title: HepG2 1

description: ucRNA profiling from HepG2 cells


title: mouse_urogenital_sinus_mesenchyme_rep5


title: mouse_urogenital_sinus_mesenchyme_rep2


title: mouse_dermis_rep2


title: mouse_dermis_rep3


title: mouse_epidermis_rep3


title: mouse_urogenital_sinus_epithelium_rep1

source_name: Total RNA of mouse urogenital sinus epithelium


title: mouse_urogenital_sinus_epithelium_rep5


title: mouse_urogenital_sinus_epithelium_rep4


title: mouse_dermis_rep4


title: mouse_urogenital_sinus_epithelium_rep3


title: mouse_epidermis_rep1


title: mouse_epidermis_rep4


title: mouse_urogenital_sinus_mesenchyme_rep3


title: mouse_urogenital_sinus_epithelium_rep6


title: mouse_urogenital_sinus_epithelium_rep2


title: 8915

status: Public on Aug 20 2010

first: Hongjuan

last: Zhao

address: 300 Pasteur Drive

department: Urology, Edwards Building R116

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-06/53383.gif

hyb_protocol: not provided

scan_protocol: Scanner Model: GenePix 4000A

source_name: Stratagene Universal Human Reference RNA

label_protocol: not provided

extract_protocol: not provided

tag: reference

source_name: 8915 Total RNA

value: Kidney, tumor tissue

tag: stage of clear cell renal cell carcinoma

value: V


title: 9799

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51699.gif

value: Stratagene Universal Human Reference RNA

source_name: 9799 Total RNA

value: cc


title: 8910

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-06/53389.gif

source_name: 8910 Total RNA


title: 9715

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-10/45571.gif

source_name: 9715 Total RNA

value: ch


title: 9112

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51684.gif

source_name: 9112 Total RNA

value: II


title: 9918

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51710.gif

source_name: 9918 Total RNA


title: 8611

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-06/53362.gif

source_name: 8611 Total RNA


title: 9702

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-10/45566.gif

source_name: 9702 Total RNA


title: 8704

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-06/53382.gif

source_name: 8704 Total RNA


title: 8621

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-06/53363.gif

source_name: 8621 Total RNA


title: 0239

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-09/44860.gif

source_name: Stratagene universal RNA

source_name: 0239 total RNA


title: 9722

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51696.gif

source_name: 9722 Total RNA


title: 0025

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-09/44625.gif

source_name: 0025 total RNA


title: 9925

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51712.gif

source_name: 9925 Total RNA


title: 9103

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-06/53374.gif

source_name: 9103 Total RNA

value: IV


title: 8818

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49612.gif

source_name: Stratagene Universal Human Reference RNA

source_name: 8818 Total RNA


title: 0018

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-07/53626.gif

source_name: 0018 Total RNA


title: 0206

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51667.gif

source_name: 0206 Total RNA


title: 8820

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49482.gif

source_name: 8820 Total RNA


title: 9126

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49316.gif

source_name: 9126 Total RNA


title: 0019

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-09/44623.gif

source_name: 0019 total RNA


title: 0235

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-08/44126.gif

source_name: 0235 Total RNA


title: 8718

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49622.gif

source_name: 8718 Total RNA


title: BMP-2 treated for 1 h at 25ng/ml rep2

status: Public on Aug 15 2010

first: Qiaoran

last: Xi

address: 1275, York ave.

department: Cancer biology and genetics

institute: MSKCC

description: E14Tg2A.4 cells were treated with BMP-2 (25ng/ml) for 1 hour.

hyb_protocol: 10 ug cRNA were hybridized to the affymetrix MOE 430 2.0 chips

scan_protocol: genechips were scanned using the Hewiett-Packard genearray scanner

data_processing: MAS 5.0

source_name: Mouse Embryonic stem cells(E14Tg2A.4)

label_protocol: biotinylated cRNA were prepared from 10ug of total RNA using the standard labeling techniques for affymetrix chips

extract_protocol: USB total RNA extraction kit


title: 9312

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49474.gif

source_name: 9312 Total RNA

value: I


title: 8726

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-03/50174.gif

source_name: 8726 Total RNA


title: Untreated control rep2

description: E14Tg2A.4 cells were untreated.


title: Pre-vitellogenic - 5

status: Public on May 01 2010

city: Vicksburg

first: Natalia

last: Vinas

state: MS

address: 3909 Halls Ferry Rd

institute: Mississippi State University

description: Data were resolved from microarray images using Agilent Feature Extraction software (Agilent Technologies, Palo Alto, CA).

hyb_protocol: The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturer’s protocol and recommendations.

scan_protocol: An Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc.) was used to scan microarray images at 5 μm resolution.

data_processing: Microarray image processing and data pre-processing were performed Agilent's Feature Extraction software v 9.5 following the manufacturer’s protocol and recommendations.

organism: Pimephales promelas

source_name: Pre-vitellogenic ovary from fathead minnow

label_protocol: cDNA synthesis, cRNA labelling, amplification and hybridization were performed following the manufacturer's kits and protocols (Quick Amp Labeling kit; Agilent, Palo Alto, CA).

growth_protocol: At the time of sample collection, fish were humanely killed in a buffered (200 mg Na2HCO3/L) solution tricaine methanesulfonate (MS-222; Finquel; Argent, Redmond WA, USA).  Gentle pressure was applied to the abdomen of each fish.  If eggs were released, the fish was noted to contain ovulated eggs, and the expressed oocytes were collected in a microcentrifuge tube and snap-frozen in liquid nitrogen.  Blood was collected from the caudal peduncle using heparinized microcapillary tubes, and plasma was separated by centrifugation and subsequently stored at -80oC.  Ovaries were removed and weighed. Tissue was transferred to a labeled microcentrifuge tube and snap-frozen in liquid nitrogen, then stored at -80o C until extracted.

extract_protocol: Total RNA was isolated from the ovary or expelled oocyte (egg) samples using RNeasy kits (Qiagen, Valencia, CA, USA).  RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and quantity was determined using a Nanodrop® ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).  Total RNA was stored at -80oC until analyzed.

tag: tissue

value: Pre-vitellogenic ovary


title: 8827-new

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-05/51677.gif

source_name: 8827 Total RNA


title: 9208

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-10/45439.gif

source_name: 9208 Total RNA


title: 9402

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2003-10/45737.gif

source_name: 9402 Total RNA


title: 9405

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49752.gif

source_name: 9405 Total RNA


title: 8913

description:
Simple annotation: Kidney, tumor tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-02/49611.gif

source_name: Stratagene Universal Human Reference RNA

source_name: 8913 Total RNA


title: Atretic - 7

source_name: Atretic ovary from fathead minnow

value: Atretic ovary


title: BMP-2 treated for 1 h at 25ng/ml rep1

description: E14Tg2A.4 cells were treated with BMP-2 at 25ng/ml for 1 hour


title: Vitellogenic - 3

source_name: Vitellogenic ovary from fathead minnow

value: Vitellogenic ovary


title: Mature ovary - 6

source_name: Mature ovary from fathead minnow

value: Mature ovary


title: Mature ovary - 5


title: Vitellogenic - 7


title: Atretic - 6


title: Vitellogenic - 1


title: Pre-vitellogenic - 2


title: Vitellogenic - 2


title: Vitellogenic - 5


title: Ovulated eggs - 2

source_name: Ovulated eggs from fathead minnow

value: Ovulated eggs


title: Ovulated eggs - 1


title: Pre-vitellogenic - 3


title: Vitellogenic - 4


title: Ovulated eggs - 4


title: Vitellogenic - 6


title: Pre-vitellogenic - 4


title: Pre-vitellogenic - 6


title: Mature ovary - 3


title: Pre-vitellogenic - 1


title: Mature ovary - 4


title: Ovulated eggs - 3


title: Atretic - 5


title: Mature ovary - 1


title: Mature ovary - 2


title: Atretic - 4


title: Atretic - 3


title: Atretic - 1


title: Atretic - 2