title: NSCB_CGH_385K_290_WA01

status: Public on Jan 28 2010

city: Madison

first: Melodee

last: Patterson

state: WI

address: 510 Charmany Dr Ste 58 & 59

institute: WiCell

description: This CGH experiment is designed to detect deletion, insertation, amplication, etc. in the sample relative to the reference.

hyb_protocol: 7.9.1.	Set the Roche NimbleGen Hybridization System 12 to 48°C. With the cover closed, allow at least 3 hours for the temperature to stabilize. 7.9.2.	  Hybridization Solution Master Mix per Chip 7.9.2.1.	Thaw Alignment Oligo on ice, protected from light. 7.9.2.2.	Add 11.8µl 2X Hybridization Buffer to 0.2ml PCR tube. 7.9.2.3.	Add 4.7µl Hybridization Component A. 7.9.2.4.	Add 0.5µl Alignment Oligo. Total amount of solution is 17.0µl. 7.9.2.5.	Vortex to mix. 7.9.2.6.	Briefly centrifuge samples using a tabletop centrifuge and keep on ice, protected from light. 7.9.3.	  Combine Test and Reference DNA Samples 7.9.3.1.	Quantify each sample using the NanoDrop as described in SOP-SS-001 7.9.3.2.	Combine 6µg each of test and reference labeled DNA in a PCR tube and bring to total volume of 5µl with VWR water. The combined volume of the test and reference samples must not exceed 5.0µl. Each of the labeled samples must fall within the concentration range of 2400-3400/ml. 7.9.3.2.1.	If the concentration of the labeled sample is low, dehydrate the labeled sample in the SpeedVac at high heat for 3 minutes with tube lids open. Monitor the volume of sample to prevent complete dehydration. 7.9.3.2.2.	If the concentration is still too low, reprecipitate starting from the second DNA precipitation (do NOT add 11.5ul of 5M NaCl) and rehydrate the pellet in a smaller volume of VWR water. 7.9.3.2.3.	If the concentration of either sample is greater than 3400 µg/ml, dilute with VWR water to achieve the acceptable concentration range. 7.9.3.3.	Add 13µl Hybridization Solution Master Mix to combined labeled DNA. 7.9.3.4.	Vortex the samples for 20 seconds and quickly centrifuge in tabletop centrifuge. Keep samples protected from light. 7.9.3.5.	Place samples in 95ºC thermocycler for 5 minutes. 7.9.3.6.	Briefly vortex and centrifuge samples. 7.9.3.7.	Quickly transfer the 0.2ml PCR tubes to the Roche NimbleGen Hybridization System 12 sample block (pre-heated to 48°C) for at least 5 	minutes before sample loading. 7.9.4.	  Prepare X1 Mixer 7.9.4.1.	Prepare X1 mixer according to NimbleGen™ Arrays User Guide (CGH Analysis v5.0). 7.9.5.	  Load Sample 7.9.5.1.	Quickly transfer labeled DNA sample from PCR tube in 48ºC sample block to a 2.0 ml tube. 7.9.5.2.	Dispense 18µl of sample into the bottom X1 mixer hole using a Gilson Microman M100 pipette. 7.9.5.3.	Dry any excess sample from X1 mixer holes using a Kimwipe. 7.9.5.4.	Seal top and bottom X1 mixer holes with mixer port seals. 7.9.5.5.	Close the bay clamp. 7.9.5.6.	Dampen humid cover with Millipore water and place over bays. Humid covers must be kept damp throughout hybridization. 7.9.5.7.	Close the Roche NimbleGen Hybridization System 12 lid. 7.9.5.8.	Turn on the Mixing Panel on the Hybridization System, set the mix mode to B, and press the mix button to start mixing. 7.9.5.9.	Confirm that the Hybridization System recognizes the slide in each occupied bay by indicator light turning green. 7.9.5.10.	Hybridize the sample to the array(s) for 40-42 hours at 48°C. 7.10.	   Sample Storage 7.10.1.	Label the remaining labeled DNA according to the following format: 7.10.2.	Store labeled DNA samples at -20C in “CGH Labeled Samples” box for up to two weeks. 7.11.	   Array Washing 7.11.1.	Turn on GenePix 4000B to warm up lasers prior to beginning wash procedure. 7.11.2.	Disassemble and wash arrays according to NimbleGen™ Arrays User Guide (CGH Analysis v5.0) with the following exceptions: 7.11.2.1.	Heat both Wash Buffer I solutions to 42ºC. 7.11.2.2.	Agitate array in Wash Buffer I for 3 minutes. 7.11.2.3.	Follow 1 minute agitation in Wash Buffer 2 with a 2 minute incubation. 7.11.3.	Perform washes protecting arrays from light and ozone. 7.11.4.	Place array in corresponding case and wrap case in aluminum foil. 7.11.4.1.	Label with CGH number. 7.11.5.	Scan arrays immediately after washing (ref: SOP-CH-309). 7.12.	   Array Storage 7.12.1.	Store scanned arrays in the dessicator.

scan_protocol: NimbleGen scan protocol

data_processing:
1. Image extraction:
NimbleScan provided by NimbleGen is used to extract signal data from image files (*.TFF). Signals from the scan for Cy3 (green channel) are saved in a file named as chipID_532.pair while signals from the scan for Cy5 (red channel) are saved in a file named as chipID_635.pair.
2. Normalization:
Signals from Cy3/Cy5 channels are transformed by logarithms to base 2 and normalized with loess or qspline (Workman et al., 2002) algorithms by R to resolve the systematic errors and bias introduced by the microarray experimental platform. The log ratios of the signals are obtained by subtracting the log transformed signals from one channel by the corresponding ones from another channel.
3. Data smoothing and break-points identification:
Ratios of the normalized data are plotted by the corresponding genome locations. The data are smoothed by an adaptive weights smoothing algorithm (GLAD R-package, Wang et al., 2005), break-points are identified by a binary segmentation algorithm (DNAcopy R-package, Olshen et al., 2004) or by the segmentation algorithm available within Nimbelgen’s Nimblescan software.
4. Data visualization:
CGH results can be saved as SignalMap GFF files and can be visualized by SignalMap provided by NimbleGen. CGH results can also be visualized by other software, such as aCGHViewer (Shankar et al., 2006), CHARMView (Myers et al., 2005), etc. However, different software requires different input format.

label_protocol:
7.7.1.	  Cy3/Cy5 Labeling
7.7.1.1.	Verify that the DNA concentration is between 25 and 30ng/ul for all test and reference samples.
7.7.1.2.	Thaw the Cy3/Cy5 random nonamers on ice, protected from light.
7.7.1.3.	Add 1µg of sonicated test DNA to 40 µl Cy3 dye. Bring to volume in 80 µl VWR water. Repeat for all test samples to be labeled.
7.7.1.4.	Add 1 µg of sonicated reference DNA to 40 µl Cy5 dye. Bring to volume in 80 µl VWR water. Repeat for all reference samples to be labeled.
7.7.1.5.	Vortex and quickly centrifuge test and reference DNA samples.
7.7.1.6.	Incubate samples for 10 minutes at 98ºC protecting from light.
7.7.1.7.	Rapidly transfer samples to an ice-water bath for 5 minutes protecting from light.
7.7.1.8.	Prepare 20µl of the following aha-dNTP/Klenow Master Mix for each test and reference sample.
7.7.1.8.1.	aha-dNTP/Klenow Master Mix per Sample
7.7.1.8.1.1.	Thaw all reagents and aha-dNTP/Klenow master mix on ice.
7.7.1.8.1.2.	Add 9µl VWR water to 0.2ml PCR tube.
7.7.1.8.1.3.	Add 10µl 50X aha-dNTP Mix.
7.7.1.8.1.4.	Add 1µl Klenow (50U/µl).  DO NOT vortex. Total amount of solution is 20µl.
7.7.1.9.	Add 20μl of the aha-dNTP/Klenow Master Mix to each of the cooled samples. Keep on ice.
7.7.1.10.	Pipette to mix (15 times). DO NOT vortex.
7.7.1.11.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.1.12.	Incubate the samples for 2 hours 15 minutes at 37°C in thermocycler protected from light.
7.7.1.13.	Remove samples from thermocycler, keep at room temperature. Heat thermocycler to 98ºC. Place samples back into thermocycler and incubate for 10 minutes at 98ºC.
7.7.1.14.	Rapidly transfer samples to an ice-water bath for 5 minutes protecting samples from light.
7.7.1.15.	Add 1 µl of Klenow (50U/µl) to each sample.
7.7.1.16.	Pipette to mix (15 times). DO NOT vortex.
7.7.1.17.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.1.18.	Place samples in thermocycler at 37ºC for overnight amplification.
7.7.2.	  First DNA Precipitation
7.7.2.1.	Set Roche Nimblegen Hybridization System 12 to 48ºC if proceeding to hybridization in the same day to allow for at least 3 hours of stabilization.
7.7.2.2.	Cool centrifuge to 4ºC.
7.7.2.3.	Add 10µl of 0.5M EDTA to each sample to stop the overnight amplification reaction.
7.7.2.4.	Add 11.5 µl of 5M NaCl to each sample, then vortex for 10 seconds.
7.7.2.5.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.2.6.	Transfer each sample into a 1.5ml tube containing 200 µl of isopropanol.
7.7.2.7.	Vortex samples for 20 seconds.
7.7.2.8.	Incubate at room temperature for 10 minutes, protected from light.
7.7.2.9.	Centrifuge at 13,000 rpm 4ºC for 10 minutes.
7.7.2.10.	Aspirate supernatant.
7.7.2.11.	Rinse pellet with 500µl of 80% ice-cold ethanol. As you rinse, gently wash pellet from tube wall.
7.7.2.12.	Centrifuge at 13,000 rpm 4ºC for 2 minutes.
7.7.2.13.	Aspirate supernatant.
7.7.2.14.	Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried.
7.7.2.15.	Proceed to Alexa Fluor Labeling or store labeled samples at -20°C, protected from light.
7.7.2.15.1.	Frozen samples are stable for up to 2 weeks at -20ºC.
7.7.3.	   Alexa Fluor Labeling
7.7.3.1.	Thaw one vial of Alexa Fluor 555 dye per Cy3-labeled test DNA sample on ice. Keep protected from light.
7.7.3.2.	Thaw one vial of Alexa Fluor 647 dye per Cy5-labeled reference DNA sample on ice. Keep protected from light.
7.7.3.3.	Thaw Alexa Fluor Labeling Buffer on ice.
7.7.3.4.	Add 5 µl VWR water to each DNA sample.
7.7.3.5.	Place samples in thermocycler at 42ºC for 5 minutes.
7.7.3.6.	Add 3 µl Labeling Buffer to each DNA sample.
7.7.3.7.	Vortex and quickly centrifuge DNA samples.
7.7.3.8.	Add 2 µl DMSO to one of the vials of Alexa Fluor dye (either 555 or 647).
7.7.3.9.	Vortex for 10 seconds to dissolve Alexa Fluor dye.
7.7.3.10.	Immediately add 8 µl of corresponding DNA to the dissolved dye.
7.7.3.11.	Pipette up and down to further dissolve dye pellet, replace lid and vortex briefly to mix.
7.7.3.12.	Incubate at room temperature, protected from light for 1 hour.
7.7.3.13.	Repeat steps 7.7.3.8.-7.7.3.12 for each Cy-labeled DNA to be labeled with Alexa Fluor dyes.
7.7.4.	   Second DNA Precipitation
7.7.4.1.	Add 100 µl VWR water to each labeled DNA sample.
7.7.4.2.	Add 11.5 µl of 5M NaCl to each sample.
7.7.4.3.	Vortex for 10 seconds.
7.7.4.4.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.4.5.	Transfer each sample into 1.5ml tubes containing 200 µl each of isopropanol.
7.7.4.6.	Vortex samples for 20 seconds.
7.7.4.7.	Incubate at room temperature for 10 minutes, protected from light.
7.7.4.8.	Centrifuge at 13,000 rpm 4ºC for 10 minutes.
7.7.4.9.	Aspirate supernatant.
7.7.4.10.	Add 500µl of 80% ice-cold ethanol to the pellet and gently wash pellet from tube wall by pipetting.
7.7.4.11.	Centrifuge at 13,000 rpm 4ºC for 2 minutes.
7.7.4.12.	Aspirate supernatant.
7.7.4.13.	Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried.
7.7.4.14.	Proceed to ‘Specific Activity Calculation’ or store labeled samples at -20°C, protected from light. Frozen samples are stable for up to 2 weeks at -20ºC.

extract_protocol:
1. Determine When to Harvest hES cells for DNA Extraction
A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity
required.
B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield.
C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells). One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage.
2. Trypsinize the Cells
A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment.
B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate.
C) Return the plate to the 37C incubator for 2-3 minutes.
D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope. Proceed to next step when colony edges begin to lift off the plate.
E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps.
F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution.
G) Gently pipet the solution up and down to mix thoroughly.
H) Centrifuge the tube for 5 minutes at 1000 RPM.
3. RNA Digestion
A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended.
B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing.
C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes.
4. Protein Digestion
A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by
vortexing. It is important that the solution is immediately and thoroughly mixed by
vortexing and pipetting to yield a homogenous solution.
NOTE: Do not add proteinase K directly to buffer AL.
B) Incubate the cell solution in a 70°C waterbath for 10 minutes.
5. DNA Precipitation and Separation of Cellular Components
A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing.
B) A white precipitate may form on addition of ethanol. This precipitate does not interfere with the DNeasy procedure.
C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 minute. Discard flowthrough and collection tube.
NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload
the spin column. The maximum number of hES cells that should be used in a DNA
extraction is 5x106 (normally, 2 wells of a 6-well plate). For larger amounts of starting material use multiple spin columns.
6. Wash the DNA
A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1.
B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube.
C)Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2.
D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube.
NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since
residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.
E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube.
7. Elution
A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane. Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute.
NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield.
B) Repeat elution step as described in 7 A).
NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step.
NOTE: More than 200μl should not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy spin column will come into contact with the elute.

tag: cell type

value: human embryonic stem (hES)

tag: cell line


title: Retina ko/wt rep1

status: Public on Jan 24 2010

type: RNA

city: Illkirch/Strasbourg

first: Wolfgang

last: Raffelsberger

address: 1 rue Laurent Fries

department: DBGS

country: France

institute: IGBMC

description: retina free of retinal pigment epithelium (RPE)

hyb_protocol: Following fragmentation, cRNA was hybridized on the GeneChip Array and the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 according to the standard Affymetrix protocol (Expression Analysis Technical Manual Rev. 2, 2005-2007, Affymetrix).

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

data_processing: RMA

label: biotin

molecule: total RNA

organism: Mus musculus

source_name: retina at age PN40

label_protocol: Biotinylated cRNA was prepared from the total RNA according to the Affymetrix protocol (Expression Analysis Technical Manual Rev. 2, 2005-2007, Affymetrix)

growth_protocol: Animals were grown until post-natal day 40.

extract_protocol: Retinal tissue was dissected free of retinal pigment epithelium (RPE) and placed in guanidine HCL.  The samples  were subsequently disrupted and homogenized using a rotor–stator  (Polytron PT2100).  Total RNA used for microarray studies was purified  from a caesium chloride gradient produced by high-speed  ultra-centrifugation.

treatment_protocol: Animals were housed under a 12 h light/12 h dark cycle and ad-lib access to food and water. Animals were sacrificed by cervical dislocation.

tag: disease state

value: heterozygous Nxnl1 null

tag: strain

value: BALB/c AJ


title: Retina wt/wt rep2

value: wild type


title: NSCB_CGH_385K_268_TE03

type: genomic

country: USA

molecule: genomic DNA

organism: Homo sapiens

label_protocol:
Labeling
7.7.1.	  Cy3/Cy5 Labeling
7.7.1.1.	Verify that the DNA concentration is between 25 and 30ng/ul for all test and reference samples.
7.7.1.2.	Thaw the Cy3/Cy5 random nonamers on ice, protected from light.
7.7.1.3.	Add 1µg of sonicated test DNA to 40 µl Cy3 dye. Bring to volume in 80 µl VWR water. Repeat for all test samples to be labeled.
7.7.1.4.	Add 1 µg of sonicated reference DNA to 40 µl Cy5 dye. Bring to volume in 80 µl VWR water. Repeat for all reference samples to be labeled.
7.7.1.5.	Vortex and quickly centrifuge test and reference DNA samples.
7.7.1.6.	Incubate samples for 10 minutes at 98ºC protecting from light.
7.7.1.7.	Rapidly transfer samples to an ice-water bath for 5 minutes protecting from light.
7.7.1.8.	Prepare 20µl of the following aha-dNTP/Klenow Master Mix for each test and reference sample.
7.7.1.8.1.	aha-dNTP/Klenow Master Mix per Sample
7.7.1.8.1.1.	Thaw all reagents and aha-dNTP/Klenow master mix on ice.
7.7.1.8.1.2.	Add 9µl VWR water to 0.2ml PCR tube.
7.7.1.8.1.3.	Add 10µl 50X aha-dNTP Mix.
7.7.1.8.1.4.	Add 1µl Klenow (50U/µl).  DO NOT vortex. Total amount of solution is 20µl.
7.7.1.9.	Add 20μl of the aha-dNTP/Klenow Master Mix to each of the cooled samples. Keep on ice.
7.7.1.10.	Pipette to mix (15 times). DO NOT vortex.
7.7.1.11.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.1.12.	Incubate the samples for 2 hours 15 minutes at 37°C in thermocycler protected from light.
7.7.1.13.	Remove samples from thermocycler, keep at room temperature. Heat thermocycler to 98ºC. Place samples back into thermocycler and incubate for 10 minutes at 98ºC.
7.7.1.14.	Rapidly transfer samples to an ice-water bath for 5 minutes protecting samples from light.
7.7.1.15.	Add 1 µl of Klenow (50U/µl) to each sample.
7.7.1.16.	Pipette to mix (15 times). DO NOT vortex.
7.7.1.17.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.1.18.	Place samples in thermocycler at 37ºC for overnight amplification.
7.7.2.	  First DNA Precipitation
7.7.2.1.	Set Roche Nimblegen Hybridization System 12 to 48ºC if proceeding to hybridization in the same day to allow for at least 3 hours of stabilization.
7.7.2.2.	Cool centrifuge to 4ºC.
7.7.2.3.	Add 10µl of 0.5M EDTA to each sample to stop the overnight amplification reaction.
7.7.2.4.	Add 11.5 µl of 5M NaCl to each sample, then vortex for 10 seconds.
7.7.2.5.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.2.6.	Transfer each sample into a 1.5ml tube containing 200 µl of isopropanol.
7.7.2.7.	Vortex samples for 20 seconds.
7.7.2.8.	Incubate at room temperature for 10 minutes, protected from light.
7.7.2.9.	Centrifuge at 13,000 rpm 4ºC for 10 minutes.
7.7.2.10.	Aspirate supernatant.
7.7.2.11.	Rinse pellet with 500µl of 80% ice-cold ethanol. As you rinse, gently wash pellet from tube wall.
7.7.2.12.	Centrifuge at 13,000 rpm 4ºC for 2 minutes.
7.7.2.13.	Aspirate supernatant.
7.7.2.14.	Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried.
7.7.2.15.	Proceed to Alexa Fluor Labeling or store labeled samples at -20°C, protected from light.
7.7.2.15.1.	Frozen samples are stable for up to 2 weeks at -20ºC.
7.7.3.	   Alexa Fluor Labeling
7.7.3.1.	Thaw one vial of Alexa Fluor 555 dye per Cy3-labeled test DNA sample on ice. Keep protected from light.
7.7.3.2.	Thaw one vial of Alexa Fluor 647 dye per Cy5-labeled reference DNA sample on ice. Keep protected from light.
7.7.3.3.	Thaw Alexa Fluor Labeling Buffer on ice.
7.7.3.4.	Add 5 µl VWR water to each DNA sample.
7.7.3.5.	Place samples in thermocycler at 42ºC for 5 minutes.
7.7.3.6.	Add 3 µl Labeling Buffer to each DNA sample.
7.7.3.7.	Vortex and quickly centrifuge DNA samples.
7.7.3.8.	Add 2 µl DMSO to one of the vials of Alexa Fluor dye (either 555 or 647).
7.7.3.9.	Vortex for 10 seconds to dissolve Alexa Fluor dye.
7.7.3.10.	Immediately add 8 µl of corresponding DNA to the dissolved dye.
7.7.3.11.	Pipette up and down to further dissolve dye pellet, replace lid and vortex briefly to mix.
7.7.3.12.	Incubate at room temperature, protected from light for 1 hour.
7.7.3.13.	Repeat steps 7.7.3.8.-7.7.3.12 for each Cy-labeled DNA to be labeled with Alexa Fluor dyes.
7.7.4.	   Second DNA Precipitation
7.7.4.1.	Add 100 µl VWR water to each labeled DNA sample.
7.7.4.2.	Add 11.5 µl of 5M NaCl to each sample.
7.7.4.3.	Vortex for 10 seconds.
7.7.4.4.	Briefly centrifuge samples using a tabletop centrifuge.
7.7.4.5.	Transfer each sample into 1.5ml tubes containing 200 µl each of isopropanol.
7.7.4.6.	Vortex samples for 20 seconds.
7.7.4.7.	Incubate at room temperature for 10 minutes, protected from light.
7.7.4.8.	Centrifuge at 13,000 rpm 4ºC for 10 minutes.
7.7.4.9.	Aspirate supernatant.
7.7.4.10.	Add 500µl of 80% ice-cold ethanol to the pellet and gently wash pellet from tube wall by pipetting.
7.7.4.11.	Centrifuge at 13,000 rpm 4ºC for 2 minutes.
7.7.4.12.	Aspirate supernatant.
7.7.4.13.	Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried.
7.7.4.14.	Proceed to ‘Specific Activity Calculation’ or store labeled samples at -20°C, protected from light. Frozen samples are stable for up to 2 weeks at -20ºC.


title: NSCB_CGH_385K_217_WA09


title: NSCB_CGH_385K_284_WA09


title: NSCB_CGH_385K_269_ES02


title: Retina ko/ko rep3

value: homozygous Nxnl1 null


title: NSCB_CGH_385K_286_TE03


title: NSCB_CGH_385K_267_WA09


title: NSCB_CGH_385K_289_SA01


title: NSCB_CGH_385K_283_WA01


title: A435_2_LPS_Luteolin

status: Public on Jan 19 2010

city: Regensburg

first: Thomas

last: Langmann

address: Franz-Josef-Strauss-Allee 11

department: University of Regensburg

country: Germany

institute: Institute of Human Genetics

description: Gene expression data from BV-2 cells treated with 50 ng/ml LPS + 50 µM Luteolin

hyb_protocol: The labeling assay yielded labeled single-stranded DNA that was fragmented and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays (expression arrays) according to manufacturer’s protocols at the KFB Regensburg

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

label: Biotin

label_protocol: Biotinylated sense-strand DNA for hybridization to Affymetrix expression arrays was prepared with the Affymetrix GeneChip whole transcript sense target labeling assay, using 300ng total RNA and standard method according to the manufacturer’s protocols (Affymetrix, GeneChip WT terminal labelling kit 900671).

growth_protocol: BV-2 cells were cultured as described in Blasi et al. J Neuroimmunol. 1990 May;27(2-3):229-37.

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

treatment_protocol: BV-2 cells were left untreated or stimulated for 24h with 50 µM Luteolin, 50 ng/ml LPS, or 50 µM Luteolin + 50 ng/ml LPS

tag: agent

value: LPS and luteolin


title: NSCB_CGH_385K_274_WA01


title: NSCB_CGH_385K_263_WA09


title: A435_1_LPS_Luteolin


title: NSCB_CGH_385K_195_WA09


title: Retina wt/wt rep1


title: NSCB_CGH_385K_259_WA09


title: A435_1_LPS

description: Gene expression data from BV-2 cells treated with 50 ng/ml LPS

value: LPS


title: A435_2_LPS


title: PAXgeneBlood_PreTreatment_Residual Inflammation (RI)_Training set_rep1_RI7

status: Public on Mar 09 2010

city: Yokohama

first: Motohiko

last: Tanino

state: Kanagawa

address: 1-1-43, Suehirocho, Tsurumi-ku

country: Japan

institute: DNA Chip Research Inc.

description: Gene expression of whole blood cells from Rheumatoid Arthritis patients before infliximab treatment

hyb_protocol: Hybridization was performed for 17 hrs at 65°C.

scan_protocol: Arrays were scanned on an Agilent DNA microarray scanner.

data_processing: Image analyses, data extraction, and background subtraction were done with the standard procedures of Agilent Feature Extraction Software Ver.9.1.

source_name: Peripheral Blood, sampling at pre treatment with infliximab infusion, residual inflammation at 14 weeks after the treatment

label_protocol: Sample RNA was amplified and 250 ng of total RNA was labeled using Cy3.

growth_protocol: RA patients who were resistant to standard MTX treatment.

extract_protocol: Blood (2.5ml) was collected from the patient into PAXgene Blood RNA Kit (QIAGEN, Valencia, CA). Total RNA was extracted according to the manufacturer’s protocol

treatment_protocol: Right before the infusion of Infliximab.

tag: tissue

value: whole blood

tag: gender

value: female

tag: age

tag: serum crp level at 14 weeks (mg/dl)


title: A435_3_LPS_Luteolin


title: A435_1_Luteolin

description: Gene expression data from BV-2 cells treated with 50 µM Luteolin

value: Luteolin


title: A435_3_LPS


title: NSCB_CGH_385K_288_SA01


title: A435_2_Luteolin


title: A435_3_Luteolin


title: A435_3_Control

description: Gene expression data from BV-2 cells

value: Control


title: A435_2_Control

description: Gene expression data from BV-2 cells


title: OS #3-1

status: Public on Jan 15 2010

city: Mississippi State

first: Chinling

last: Wang

state: MS

address: College of Veterinary Medicine P.O. box 6100

department: Basic Sciences

institute: Missippi State University

description:
Cy5-No NaCl
Cy3-1.2% NaCl
Aminoallyl-labelled Listeria F2365 cDNA microarray slides (Version 3) kindly provided by the Pathogen Functional Genomic Research Centre (PFGRC) were used. The slides consist of 70-mer oligonucleotides representing 2847 open reading frames from L. monocytogenes strains 4b F2365 (GenBank Accession no. AE017262).

hyb_protocol: The cDNA labelled with cy3 or cy5 was resuspended in 0.1 M sodium carbonate buffer (pH 9.3). About 5.8 ug of each cDNA were hybridized, incubated for 1h at RT, and resuspended in 50 ul hybridization buffer (40% formamide, 5X sodium chloride/sodium citrate buffer, 0.1% SDS, and 0.6 ug/ul salmon sperm DNA).  The resuspended sample was loaded on a microarray slide. The slide was incubated for 16 h at 42°C, and then washed with low, medium, high and stringency buffer containing 0.1 mM DTT.

scan_protocol: The hybridized slides were scanned at 10 um resolutions within the range of photomultiplier tube between 650 and 750 nm on two channels, cy3 and cy5 dyes using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA).

data_processing: TM4 package developed by TIGR was used for data analysis. The signal intensity of spots on the slides was adjusted and quantified by background subtraction using the Spotfinder. Data LOWESS normalization was performed using the Ginkgo. Magnolia (v.1.2) was used to produce SOFT file format.

organism: Listeria monocytogenes

source_name: total_RNA from Listeria monocytogenes F2365 labeled with cy5.

label_protocol: To synthesize cDNA, 2.5 ug of the RNA samples were reversely transcribed. cDNA was synthesized using 2 ul of Smart reverse transcriptase (Clontech, Palo Alto, CA) and 1 ul of random hexamers in the presence of 0.1 M dithiothreitol (DTT) [Invitrogen], 25 mM dNTP with 3:2 ratio of aminoallyl (aa)-dUTP and dTTP (Ambion, Austin, TX). After cDNA synthesis, the cDNA was purified and unincorpotated aa-dUTP was removed using a MinElute PCR purification kit (Qiagen). The purified cDNA resuspended in 0.1 M sodium carbonate buffer (pH 9.3) was labelled with cy5.

growth_protocol: 25 ul of the bacterial culture (about 2.9 x 10^8 CFU/ml) was inoculated in 5 ml of a fresh BHI medium without 1.2% NaCl and incubated at 37°C for 6 h in a MaxQ™ 4000 incubating and orbital shaker (Barnstead/Lab-Line, Dubuque, IA) at 180 rpm till the O.D. reached to A600 ≈ 0.7.

extract_protocol: Total RNA was extracted from the bacterial pellet using Trizol™ (Invitrogen, Carlsbad, CA) and purified  with the RNeasy Mini Kit (Qiagen).

treatment_protocol: add: No NaCl

source_name: total_RNA from Listeria monocytogenes F2365 labeled with cy3.

label_protocol: To synthesize cDNA, 2.5 ug of the RNA samples were reversely transcribed. cDNA was synthesized using 2 ul of Smart reverse transcriptase (Clontech, Palo Alto, CA) and 1 ul of random hexamers in the presence of 0.1 M dithiothreitol (DTT) [Invitrogen], 25 mM dNTP with 3:2 ratio of aminoallyl (aa)-dUTP and dTTP (Ambion, Austin, TX). After cDNA synthesis, the cDNA was purified and unincorpotated aa-dUTP was removed using a MinElute PCR purification kit (Qiagen). The purified cDNA resuspended in 0.1 M sodium carbonate buffer (pH 9.3) was labelled with cy3.

growth_protocol: 25 ul of the bacterial culture (about 2.9 x 10^8 CFU/ml) was inoculated in 5 ml of a fresh BHI medium added with 1.2% NaCl and incubated at 37°C for 6 h in a MaxQ™ 4000 incubating and orbital shaker (Barnstead/Lab-Line, Dubuque, IA) at 180 rpm till the O.D. reached to A600 ≈ 0.7.

treatment_protocol: add: 1.2% NaCl


title: OS #3-2


title: OS #3-4

description:
Cy3-No NaCl
Cy5-1.2% NaCl
Aminoallyl-labelled Listeria F2365 cDNA microarray slides (Version 3) kindly provided by the Pathogen Functional Genomic Research Centre (PFGRC) were used. The slides consist of 70-mer oligonucleotides representing 2847 open reading frames from L. monocytogenes strains 4b F2365 (GenBank Accession no. AE017262).


title: A435_1_Control

description: Gene expression data from BV-2 cells


title: OS #2-4


title: OS #2-2

label_protocol: To synthesize cDNA, 2.5 ug of the RNA samples were reversely transcribed cDNA was synthesized using 2 ul of Smart reverse transcriptase (Clontech, Palo Alto, CA) and 1 ul of random hexamers in the presence of 0.1 M dithiothreitol (DTT) [Invitrogen], 25 mM dNTP with 3:2 ratio of aminoallyl (aa)-dUTP and dTTP (Ambion, Austin, TX). After cDNA synthesis, the cDNA was purified and unincorpotated aa-dUTP was removed using a MinElute PCR purification kit (Qiagen). The purified cDNA resuspended in 0.1 M sodium carbonate buffer (pH 9.3) was labelled with cy5.


title: OS #2-1


title: Biological sample 3 Replicate 1

status: Public on Jan 07 2010

city: Storrs

first: Steven

middle: J.

last: Geary

state: CT

address: 61 N. Eagleville Rd

department: Pathobiology and Veterinary Science

institute: University of Connecticut

description: Mycoplasma gallisepticum stains Rlow vs. F in vitro transcriptomes.  Biological replicate 3 of 3, technical replicate 1 of 2.  Rlow Cy3/F Cy5

hyb_protocol: Samples were hybridized to oligo DNA microarrays in a humidified hybridization chamber overnight at 42 degrees C with shaking at 70RPM.  Slides were washed with decreasing contrations of SSC buffer for 5 min each and centrifuged at 500RPM for 5 min.

scan_protocol: Slides were scanned in a GenePix 4000B array scanner such that a channel ratio of about 1 was achieved and PMT gain was increased until a few features contained pixels that reached saturation.

data_processing: Images were quantified using Aquity software to generate the raw data set.  635/532 ratios were generated after local background subtraction from each channel

organism: Mycoplasmoides gallisepticum

source_name: Rlow

label_protocol: 100ug mRNA was reverse transcribed using the Amino Allyl kit from Ambion per the manufacturer's instructions.  Biological samples were split into two 50ug replicates and one was labeled with Cy3 and the other Cy5.  Strains, labeled with opposite dyes, were mixed, column purified, and resuspended in hybridization buffer.

growth_protocol: Mycoplasma gallisepticum strains Rlow and F cells grown to mid-log phase in complete Hayflick's growth medium, placed on ice for 15 min. and pelleted via centrifugation at 10000RPM.

extract_protocol: Total RNA extracted using the Trizol Reagent per the manufacturer's instructions.  RNA was DNase treated using the Ambion DNA free reagent per the manufacturer's instructions. Samples were quantified using OD260. Convenctional PCR was conducted on aliquot of DNase treated RNA using 16s rRNA primers to ensure there was no DNA contamination.

value: Rlow

source_name: F

value: F


title: OS #1-4

label_protocol: To synthesize cDNA, 2.5 ug of the RNA samples were reversely transcribed cDNA was synthesized using 2 ul of Smart reverse transcriptase (Clontech, Palo Alto, CA) and 1 ul of random hexamers in the presence of 0.1 M dithiothreitol (DTT) [Invitrogen], 25 mM dNTP with 3:2 ratio of aminoallyl (aa)-dUTP and dTTP (Ambion, Austin, TX). After cDNA synthesis, the cDNA was purified and unincorpotated aa-dUTP was removed using a MinElute PCR purification kit (Qiagen). The purified cDNA resuspended in 0.1 M sodium carbonate buffer (pH 9.3) was labelled with cy3.

treatment_protocol: add: 1.2 NaCl


title: OS #2-3

source_name: total_RNA from Listeria monocytogenes F2365 labeled with Cy5.


title: Biological sample 1 Replicate 1

description: Mycoplasma gallisepticum stains Rlow vs. F in vitro transcriptomes.  Biological replicate 1 of 3, technical replicate 1 of 2.  Rlow Cy3/F Cy5


title: Biological sample 3 Replicate 2

description: Mycoplasma gallisepticum stains Rlow vs. F in vitro transcriptomes.  Biological replicate 3 of 3, technical replicate 2 of 2.  Rlow Cy5/F Cy3


title: Biological sample 2 Replicate 1

description: Mycoplasma gallisepticum stains Rlow vs. F in vitro transcriptomes.  Biological replicate 2 of 3, technical replicate 1 of 2.  Rlow Cy3/F Cy5


title: OS #3-3


title: OS #1-1


title: OS #1-3


title: OS #1-2


title: Biological sample 2 Replicate 2

description: Mycoplasma gallisepticum stains Rlow vs. F in vitro transcriptomes.  Biological replicate 2 of 3, technical replicate 2 of 2.  Rlow Cy5/F Cy3


title: Biological sample 1 Replicate 2

description: Mycoplasma gallisepticum stains Rlow vs. F in vitro transcriptomes.  Biological replicate 1 of 3, technical replicate 2 of 2.  Rlow Cy5/F Cy3


title: CEPI_IV_542

status: Public on Dec 31 2009

city: Atlanta

first: Nathan

last: Bowen

state: GA

address: 223 James P Brawley Drive

department: Center for Cancer Research and Therapeutic Development

institute: Clark Atlanta University

description: Serous ovarian tumor tissues were placed in cryotubes and immediately (<1 minute) frozen in liquid nitrogen following resection.

hyb_protocol: Labeled mRNA was hybridized to GeneChip Human Genome U133 Plus 2.0 Arrays in the GeneChip Hybridization oven 640, further processed with the GeneChip Fluidics Station 450 and scanned with the GeneChip Scanner.

scan_protocol: as per manufacturer's protocol

data_processing: MAS 5.0 via Affymetrix Expression Console (TM) Software Version 1.1

source_name: Laser Capture Microdissected Serous Ovarian Cancer Epithelia

label_protocol: Total RNA from the above extractions was processed using the RiboAmp OA or HS kit (Arcturus) in conjunction with the IVT Labeling Kit from Affymetrix, to produce an amplified, biotin-labeled mRNA suitable for hybridizing to GeneChip Probe Arrays (Affymetrix).

extract_protocol: RNA was isolated with Trizol (Invitrogen) and purified with PicoPure RNA Isolation Kit (Arcturus). RNA quality was verified on the Bioanalyzer RNA Pico Chip (Agilent Technologies).

treatment_protocol: Following pathological verification, a serous papillary epithelial cancer sample was embedded in cryomatrix (Shandon).  Seven micron frozen sections were cut and attached to uncharged microscope slides for each sample.  Immediately following dehydration and staining (HistoGene, LCM Frozen Section Staining Kit, Arcturus), slides were processed in an Autopix‘ instrument (Arcturus) for laser capture microdissection (LCM) of cells to CapSure Macro LCM Caps.  Approximately 30,000 epithelia were collected from each cancer sample.

value: ovarian adenocarcinoma

tag: specimen

value: ovarian cancer epithelia cells


title: CEPI_IV_489


title: CEPI_IIIc_528


title: CEPI_IIIc_588


title: CEPI_IIIc_369


title: CEPI_IIIc_242


title: CEPI_IIb_413


title: CEPI_II_367


title: CEPI_Ic_336


title: CEPI_IIIc_229


title: OSE_440

description: Ovarian surface epithelia brushing from ovaries described by pathology as within normal limits.

hyb_protocol: as per manufacturer's protocol

source_name: Ovarian Surface Epithelia

label_protocol: Affymetrix IVT Labeling Kit

treatment_protocol: Normal ovarian surface epithelial (OSE) cells were collected from ovaries at time of surgery using a Cytobrush ® Plus (Medscand) and immediately stabilized in RNAlater (Ambion) and stored at –20 degrees C.

value: normal

value: Ovarian Surface Epithelia


title: OSE_470


title: OSE_475


title: OSE_463


title: NSCB_CGH_385K_287_ES02

value: human embryonic stem (hES)


title: OSE_448


title: CEPI_III_183


title: OSE_567


title: OSE_434


title: OSE_541


title: OSE_552


title: CEPI_Ic_317


title: OSE_437


title: OSE_452


title: OSE_563


title: Low Salt Diet project_LSD dog heart sample LSD2

status: Public on Dec 22 2009

city: Valhalla

first: Caroline

last: Ojaimi

state: NY

institute: New York Medical College

description: RNA extracted from the LV of dog hearts

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: GCOS/ExpressionStat 5.0

organism: Canis lupus familiaris

source_name: LSD dog heart

label_protocol: Affymetrix labeling procedure

extract_protocol: RNA extracted using Trizol reagents

value: heart

tag: protocol

value: low salt diet

value: male


title: Low Salt Diet project_LSD dog heart sample LSD4

description: RNA extracted from the LV of dog hearts


title: Low Salt Diet project_control dog heart sample DC1

description: RNA extracted from the LV of dog hearts

source_name: control dog heart

value: control


title: Melanoma cell line LB2259-MEL TNFa, biological rep1

status: Public on Dec 11 2009

city: Brussels

first: Nicolas

last: van Baren

address: 74 avenue Hippocrate, UCL7459

country: Belgium

institute: Ludwig Institute for Cancer Research

description: no additional information

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45°C on HG-U133A2.0 gene chips (Affymetrix, Santa Clara, CA), which were then washed and stained with the Fluidics Station 450 (Affymetrix)

scan_protocol: GeneChips were scanned using the Gene Chip Scanner 3000 (Affymetrix)

data_processing:
Data acquisition and processing were conducted with Affymetrix Genechips Operating Software (using Affymetrix default analysis settings and global scaling as normalization method; the trimmed mean target intensity of each array was arbitrarily set to 100), and Microsoft Excel softwares.
CEL file unavailable for this Sample (original files erased, backup files corrupt).

source_name: Melanoma cell line LB2259-MEL treated with TNFa 10 ng/ml for 72h

label_protocol: Biotin-labeled antisense cRNA was prepared from 5 μg of total RNA using the one-cycle target labeling procedure (according to the Affymetrix Geneship Expression Analysis Technical manual, 2001)

growth_protocol: The melanoma cell lines were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum, 0.55 mM L-arginine, 0.24 mM L-asparagine, 1.5 mM L-glutamine (AAG), 100 U/ml  penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 8% CO2.

extract_protocol: RNA was extracted using the guanidinium isothiocyanate / cesium chloride procedure

treatment_protocol: Melanoma cell lines were treated with either IFN-α 1,000 U/ml, IFN-γ 100 U/ml or TNF-α 10 ng/ml for 72 hours. Cells were collected at rest or after stimulation with cytokines.

value: Melanoma cell line LB2259

value: TNFa


title: Low Salt Diet project_control dog heart sample DC3

description: RNA extracted from the LV of dog hearts


title: Melanoma cell line LB2259-MEL IFNa, biological rep1

description: no additional information

source_name: Melanoma cell line LB2259-MEL treated with IFNa 1,000 U/ml for 72h

value: IFNa


title: Melanoma cell line LB2259-MEL CTRL, biological rep1

description: no additional information

source_name: Melanoma cell line LB2259-MEL Control


title: Low Salt Diet project_LSD dog heart sample LSD3

description: RNA extracted from the LV of dog hearts


title: Low Salt Diet project_LSD dog heart sample LSD1

description: RNA extracted from the LV of dog hearts


title: Melanoma cell line LB2117-MEL TNFa, biological rep1

description: no additional information

data_processing: Data acquisition and processing were conducted with Affymetrix Genechips Operating Software (using Affymetrix default analysis settings and global scaling as normalization method; the trimmed mean target intensity of each array was arbitrarily set to 100), and Microsoft Excel softwares.

source_name: Melanoma cell line LB2117-MEL treated with TNFa 10 ng/ml for 72h

value: Melanoma cell line LB2117


title: Low Salt Diet project_control dog heart sample DC4

description: RNA extracted from the LV of dog hearts


title: Melanoma cell line LB2117-MEL IFNg, biological rep1

description: no additional information

source_name: Melanoma cell line LB2117-MEL treated with IFNg 100 U/ml for 72h

value: IFNg


title: Melanoma cell line LB2117-MEL IFNa, biological rep1

description: no additional information

source_name: Melanoma cell line LB2117-MEL treated with IFNa 1,000 U/ml for 72h


title: Melanoma cell line LB2117-MEL CTRL, biological rep1

description: no additional information

source_name: Melanoma cell line LB2117-MEL Control


title: Melanoma cell line EB81-MEL TNFa, biological rep1

description: no additional information

source_name: Melanoma cell line EB81-MEL treated with TNFa 10 ng/ml for 72h

value: Melanoma cell line EB81


title: Melanoma cell line LB2259-MEL IFNg, biological rep1

description: no additional information

source_name: Melanoma cell line LB2259-MEL treated with IFNg 100 U/ml for 72h


title: Melanoma cell line EB81-MEL IFNa, biological rep1

description: no additional information

source_name: Melanoma cell line EB81-MEL treated with IFNa 1,000 U/ml for 72h


title: Melanoma cell line EB81-MEL CTRL, biological rep1

description: no additional information

source_name: Melanoma cell line EB81-MEL Control


title: Melanoma cell line DDHK62-MEL IFNg, biological rep1

description: no additional information

source_name: Melanoma cell line DDHK62-MEL treated with IFNg 100 U/ml for 72h

value: Melanoma cell line DDHK62


title: Melanoma cell line DDHK62-MEL TNFa, biological rep1

description: no additional information

source_name: Melanoma cell line DDHK62-MEL treated with TNFa 10 ng/ml for 72h


title: Melanoma cell line EB81-MEL IFNg, biological rep1

description: no additional information

source_name: Melanoma cell line EB81-MEL treated with IFNg 100 U/ml for 72h


title: Melanoma cell line DDHK62-MEL CTRL, biological rep1

description: no additional information

source_name: Melanoma cell line DDHK62-MEL Control


title: Melanoma cell line LB2586-MEL TNFa, biological rep1

description: no additional information

source_name: Melanoma cell line LB2586-MEL treated with TNFa 10 ng/ml for 72h

value: Melanoma cell line LB2586


title: Melanoma cell line LB2586-MEL IFNg, biological rep1

description: no additional information

source_name: Melanoma cell line LB2586-MEL treated with IFNg 100 U/ml for 72h


title: Melanoma cell line LB2586-MEL IFNa, biological rep1

description: no additional information

source_name: Melanoma cell line LB2586-MEL treated with IFNa 1,000 U/ml for 72h


title: Melanoma cell line LB2586-MEL CTRL, biological rep1

description: no additional information

source_name: Melanoma cell line LB2586-MEL Control


title: Melanoma cell line DDHK62-MEL IFNa, biological rep1

description: no additional information

source_name: Melanoma cell line DDHK62-MEL treated with IFNa 1,000 U/ml for 72h


title: NSCB_CGH_385K_258_SA02

status: Public on Dec 07 2009

hyb_protocol: 7.9.1. Set the Roche NimbleGen Hybridization System 12 to 48°C. With the cover closed, allow at least 3 hours for the temperature to stabilize. 7.9.2. Hybridization Solution Master Mix per Chip 7.9.2.1. Thaw Alignment Oligo on ice, protected from light. 7.9.2.2. Add 11.8µl 2X Hybridization Buffer to 0.2ml PCR tube. 7.9.2.3. Add 4.7µl Hybridization Component A. 7.9.2.4. Add 0.5µl Alignment Oligo. Total amount of solution is 17.0µl. 7.9.2.5. Vortex to mix. 7.9.2.6. Briefly centrifuge samples using a tabletop centrifuge and keep on ice, protected from light. 7.9.3. Combine Test and Reference DNA Samples 7.9.3.1. Quantify each sample using the NanoDrop as described in SOP-SS-001 7.9.3.2. Combine 6µg each of test and reference labeled DNA in a PCR tube and bring to total volume of 5µl with VWR water. The combined volume of the test and reference samples must not exceed 5.0µl. Each of the labeled samples must fall within the concentration range of 2400-3400/ml. 7.9.3.2.1. If the concentration of the labeled sample is low, dehydrate the labeled sample in the SpeedVac at high heat for 3 minutes with tube lids open. Monitor the volume of sample to prevent complete dehydration. 7.9.3.2.2. If the concentration is still too low, reprecipitate starting from the second DNA precipitation (do NOT add 11.5ul of 5M NaCl) and rehydrate the pellet in a smaller volume of VWR water. 7.9.3.2.3. If the concentration of either sample is greater than 3400 µg/ml, dilute with VWR water to achieve the acceptable concentration range. 7.9.3.3. Add 13µl Hybridization Solution Master Mix to combined labeled DNA. 7.9.3.4. Vortex the samples for 20 seconds and quickly centrifuge in tabletop centrifuge. Keep samples protected from light. 7.9.3.5. Place samples in 95ºC thermocycler for 5 minutes. 7.9.3.6. Briefly vortex and centrifuge samples. 7.9.3.7. Quickly transfer the 0.2ml PCR tubes to the Roche NimbleGen Hybridization System 12 sample block (pre-heated to 48°C) for at least 5 minutes before sample loading. 7.9.4. Prepare X1 Mixer 7.9.4.1. Prepare X1 mixer according to NimbleGen™ Arrays User Guide (CGH Analysis v5.0). 7.9.5. Load Sample 7.9.5.1. Quickly transfer labeled DNA sample from PCR tube in 48ºC sample block to a 2.0 ml tube. 7.9.5.2. Dispense 18µl of sample into the bottom X1 mixer hole using a Gilson Microman M100 pipette. 7.9.5.3. Dry any excess sample from X1 mixer holes using a Kimwipe. 7.9.5.4. Seal top and bottom X1 mixer holes with mixer port seals. 7.9.5.5. Close the bay clamp. 7.9.5.6. Dampen humid cover with Millipore water and place over bays. Humid covers must be kept damp throughout hybridization. 7.9.5.7. Close the Roche NimbleGen Hybridization System 12 lid. 7.9.5.8. Turn on the Mixing Panel on the Hybridization System, set the mix mode to B, and press the mix button to start mixing. 7.9.5.9. Confirm that the Hybridization System recognizes the slide in each occupied bay by indicator light turning green. 7.9.5.10. Hybridize the sample to the array(s) for 40-42 hours at 48°C. 7.10. Sample Storage 7.10.1. Label the remaining labeled DNA according to the following format: 7.10.2. Store labeled DNA samples at -20C in “CGH Labeled Samples” box for up to two weeks. 7.11. Array Washing 7.11.1. Turn on GenePix 4000B to warm up lasers prior to beginning wash procedure. 7.11.2. Disassemble and wash arrays according to NimbleGen™ Arrays User Guide (CGH Analysis v5.0) with the following exceptions: 7.11.2.1. Heat both Wash Buffer I solutions to 42ºC. 7.11.2.2. Agitate array in Wash Buffer I for 3 minutes. 7.11.2.3. Follow 1 minute agitation in Wash Buffer 2 with a 2 minute incubation. 7.11.3. Perform washes protecting arrays from light and ozone. 7.11.4. Place array in corresponding case and wrap case in aluminum foil. 7.11.4.1. Label with CGH number. 7.11.5. Scan arrays immediately after washing (ref: SOP-CH-309). 7.12. Array Storage 7.12.1. Store scanned arrays in the dessicator.

data_processing:
1. Image extraction:
NimbleScan provided by NimbleGen is used to extract signal data from image files
(*.TFF). Signals from the scan for Cy3 (green channel) are saved in a file named as chipID_532.pair while signals from the scan for Cy5 (red channel) are saved in a file named as chipID_635.pair.
2. Normalization:
Signals from Cy3/Cy5 channels are transformed by logarithms to base 2 and normalized with loess or qspline (Workman et al., 2002) algorithms by R to resolve the systematic errors and bias introduced by the microarray experimental platform. The log ratios of the signals are obtained by subtracting the log transformed signals from one channel by the corresponding ones from another channel.
3. Data smoothing and break-points identification:
Ratios of the normalized data are plotted by the corresponding genome locations. The data are smoothed by an adaptive weights smoothing algorithm (GLAD R-package, Wang et al., 2005), break-points are identified by a binary segmentation algorithm (DNAcopy R-package, Olshen et al., 2004) or by the segmentation algorithm available within Nimbelgen’s Nimblescan software.
4. Data visualization:
CGH results can be saved as SignalMap GFF files and can be visualized by SignalMap provided by NimbleGen. CGH results can also be visualized by other software, such as aCGHViewer (Shankar et al., 2006), CHARMView (Myers et al., 2005), etc. However, different software requires different input format.

label_protocol:
7.7.1. Cy3/Cy5 Labeling
7.7.1.1. Verify that the DNA concentration is between 25 and 30ng/ul for all test and reference samples.
7.7.1.2. Thaw the Cy3/Cy5 random nonamers on ice, protected from light.
7.7.1.3. Add 1µg of sonicated test DNA to 40 µl Cy3 dye. Bring to volume in 80 µl VWR water. Repeat for all test samples to be labeled.
7.7.1.4. Add 1 µg of sonicated reference DNA to 40 µl Cy5 dye. Bring to volume in 80 µl VWR water. Repeat for all reference samples to be labeled.
7.7.1.5. Vortex and quickly centrifuge test and reference DNA samples.
7.7.1.6. Incubate samples for 10 minutes at 98ºC protecting from light.
7.7.1.7. Rapidly transfer samples to an ice-water bath for 5 minutes protecting from light.
7.7.1.8. Prepare 20µl of the following aha-dNTP/Klenow Master Mix for each test and reference sample.
7.7.1.8.1. aha-dNTP/Klenow Master Mix per Sample
7.7.1.8.1.1. Thaw all reagents and aha-dNTP/Klenow master mix on ice.
7.7.1.8.1.2. Add 9µl VWR water to 0.2ml PCR tube.
7.7.1.8.1.3. Add 10µl 50X aha-dNTP Mix.
7.7.1.8.1.4. Add 1µl Klenow (50U/µl). DO NOT vortex. Total amount of solution is 20µl.
7.7.1.9. Add 20μl of the aha-dNTP/Klenow Master Mix to each of the cooled samples. Keep on ice.
7.7.1.10. Pipette to mix (15 times). DO NOT vortex.
7.7.1.11. Briefly centrifuge samples using a tabletop centrifuge.
7.7.1.12. Incubate the samples for 2 hours 15 minutes at 37°C in thermocycler protected from light.
7.7.1.13. Remove samples from thermocycler, keep at room temperature. Heat thermocycler to 98ºC. Place samples back into thermocycler and incubate for 10 minutes at 98ºC.
7.7.1.14. Rapidly transfer samples to an ice-water bath for 5 minutes protecting samples from light.
7.7.1.15. Add 1 µl of Klenow (50U/µl) to each sample.
7.7.1.16. Pipette to mix (15 times). DO NOT vortex.
7.7.1.17. Briefly centrifuge samples using a tabletop centrifuge.
7.7.1.18. Place samples in thermocycler at 37ºC for overnight amplification.
7.7.2. First DNA Precipitation
7.7.2.1. Set Roche Nimblegen Hybridization System 12 to 48ºC if proceeding to hybridization in the same day to allow for at least 3 hours of stabilization.
7.7.2.2. Cool centrifuge to 4ºC.
7.7.2.3. Add 10µl of 0.5M EDTA to each sample to stop the overnight amplification reaction.
7.7.2.4. Add 11.5 µl of 5M NaCl to each sample, then vortex for 10 seconds.
7.7.2.5. Briefly centrifuge samples using a tabletop centrifuge.
7.7.2.6. Transfer each sample into a 1.5ml tube containing 200 µl of isopropanol.
7.7.2.7. Vortex samples for 20 seconds.
7.7.2.8. Incubate at room temperature for 10 minutes, protected from light.
7.7.2.9. Centrifuge at 13,000 rpm 4ºC for 10 minutes.
7.7.2.10. Aspirate supernatant.
7.7.2.11. Rinse pellet with 500µl of 80% ice-cold ethanol. As you rinse, gently wash pellet from tube wall.
7.7.2.12. Centrifuge at 13,000 rpm 4ºC for 2 minutes.
7.7.2.13. Aspirate supernatant.
7.7.2.14. Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried.
7.7.2.15. Proceed to Alexa Fluor Labeling or store labeled samples at -20°C, protected from light.
7.7.2.15.1. Frozen samples are stable for up to 2 weeks at -20ºC.
7.7.3. Alexa Fluor Labeling
7.7.3.1. Thaw one vial of Alexa Fluor 555 dye per Cy3-labeled test DNA sample on ice. Keep protected from light.
7.7.3.2. Thaw one vial of Alexa Fluor 647 dye per Cy5-labeled reference DNA sample on ice. Keep protected from light.
7.7.3.3. Thaw Alexa Fluor Labeling Buffer on ice.
7.7.3.4. Add 5 µl VWR water to each DNA sample.
7.7.3.5. Place samples in thermocycler at 42ºC for 5 minutes.
7.7.3.6. Add 3 µl Labeling Buffer to each DNA sample.
7.7.3.7. Vortex and quickly centrifuge DNA samples.
7.7.3.8. Add 2 µl DMSO to one of the vials of Alexa Fluor dye (either 555 or 647).
7.7.3.9. Vortex for 10 seconds to dissolve Alexa Fluor dye.
7.7.3.10. Immediately add 8 µl of corresponding DNA to the dissolved dye.
7.7.3.11. Pipette up and down to further dissolve dye pellet, replace lid and vortex briefly to mix.
7.7.3.12. Incubate at room temperature, protected from light for 1 hour.
7.7.3.13. Repeat steps 7.7.3.8.-7.7.3.12 for each Cy-labeled DNA to be labeled with Alexa Fluor dyes.
7.7.4. Second DNA Precipitation
7.7.4.1. Add 100 µl VWR water to each labeled DNA sample.
7.7.4.2. Add 11.5 µl of 5M NaCl to each sample.
7.7.4.3. Vortex for 10 seconds.
7.7.4.4. Briefly centrifuge samples using a tabletop centrifuge.
7.7.4.5. Transfer each sample into 1.5ml tubes containing 200 µl each of isopropanol.
7.7.4.6. Vortex samples for 20 seconds.
7.7.4.7. Incubate at room temperature for 10 minutes, protected from light.
7.7.4.8. Centrifuge at 13,000 rpm 4ºC for 10 minutes.
7.7.4.9. Aspirate supernatant.
7.7.4.10. Add 500µl of 80% ice-cold ethanol to the pellet and gently wash pellet from tube wall by pipetting.
7.7.4.11. Centrifuge at 13,000 rpm 4ºC for 2 minutes.
7.7.4.12. Aspirate supernatant.
7.7.4.13. Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried.
7.7.4.14. Proceed to ‘Specific Activity Calculation’ or store labeled samples at -20°C, protected from light. Frozen samples are stable for up to 2 weeks at -20ºC.

source_name: Promega Pooled Reference


title: NSCB_CGH_385K_244_UC01


title: NSCB_CGH_385K_257_ES02


title: NSCB_CGH_385K_256_TE03

source_name: TE03 DNA391


title: NSCB_CGH_385K_255_WA09


title: NSCB_CGH_385K_250_BG03


title: NSCB_CGH_385K_254_BG03


title: NSCB_CGH_385K_203_WA09


title: NSCB_CGH_385K_245_WA09

source_name: WA09 DNA356


title: NSCB_CGH_385K_246_ES02

source_name: ES02 DNA357


title: NSCB_CGH_385K_252_WA09


title: NSCB_CGH_385K_249_ES02


title: NSCB_CGH_385K_247_TE03

source_name: TE03 DNA360


title: NSCB_CGH_385K_248_ES02


title: NSCB_CGH_385K_241_ES03


title: NSCB_CGH_390K_16267002

hyb_protocol: 1. Incubate at 95°C (in heat block), 5 min., spin briefly 2. Place at 42°C (in Maui machine), 5-15 min. 3. Prepare microarray: A)pre-heat array on Maui station B) put array into array-holder (with number facing up going in first) carefully remove glue-sticker from lid and put lid onto array C) take array w/ lid out of array-holder thoroughly attach lid using thick slide place array onto Maui station, approx. 2 min. make sure lid is completely attached D) Add 40 µl hyb solution onto microarray and allow to settle (approx. 15 sec.), dry lid thoroughly 4. Place stickers onto lid, press down on both at the same time, make sure are thoroughly attached 5. Squeeze bubbles away from center of array 6. Wet humid cover and place on Maui station 7. Incubate on Maui at 42°C overnight (17 hours) 8. Remove microarray w/lid from Maui and put in array-holder in Wash 1 (at 42°C) 9. Peel lid off chip while submerged 10. Incubate 10-15 sec. w/ gentle agitation 11. Transfer slide into a slide rack in a new container of Wash 1 (r.t.) Incubate 2 min. w/ gentle agitation 12. Transfer to Wash 2 13. Incubate 1 min. w/ gentle agitation 14. Transfer to Wash 3 15. Incubate 15 sec. w/ gentle agitation 16. Spin approx. 1 min. in Array-Go-Round (place arrays such that numbers are facing to the right when rack is placed in front part of wheel) (the rod on slide holder should be on the inside) 17. Blow off any residual liquid 18. Keep slides protected from light and scan as soon as possible

label_protocol:
1. Dry the sample in the Speed Vac set at 30°C.
2. Resuspend the sample in 4.5μl 0.1 M Na2CO3, pH 9.0.
3. Resuspend the Cy3 NHS ester in 4.5μl DMSO
4. Add Cy3 NHS ester to sample
5. Incubate at 22°C in a heat block for 1 hour. Protect the heat block and sample from light.
6. Purify the sample using a Qiaquick PCR purification Kit:
A) Add 35μl 100 mM NaOAc, pH 5.2 to the sample.
B) Add 250μl PB buffer to the sample and load the sample on the column.
C) Spin at 13.000 rpm for 1 minute and remove the flow through.
D) Add 750μl PE buffer to the column.
E) Spin at 13.000 rpm for 1 minute and remove the flow through.
F) Spin at 13.000 rpm for 1 minute.
G) Transfer the column to new tube.
H) Add 30μl H20 and incubate for 1 minute on the column.
I) Elute once by centrifuging at 13.000 rpm for 1 minute.

source_name: Human Reference Genomic DNA


title: NSCB_CGH_385K_242_WA09


title: NSCB_CGH_390K_16267302


title: NSCB_CGH_385K_167_WA01


title: NSCB_CGH_390K_16267702


title: NSCB_CGH_385K_243_TE03


title: NSCB_CGH_390K_16260002


title: NSCB_CGH_390K_16261402


title: NSCB_CGH_385K_253_ES02


title: NSCB_CGH_390K_16259102


title: NSCB_CGH_390K_16263302


title: NSCB_CGH_390K_16256802


title: NSCB_CGH_390K_16256702


title: NSCB_CGH_390K_16265902


title: NSCB_CGH_390K_16255902


title: NSCB_CGH_390K_16250502


title: NSCB_CGH_390K_16257002


title: NSCB_CGH_390K_16259402


title: NSCB_CGH_390K_16250402


title: NSCB_CGH_390K_16262402


title: NSCB_CGH_390K_16252002


title: Eset shRNA ES cells, biological replicate 1

status: Public on Nov 04 2009

city: Singapore

first: Ping

last: Yuan

address: 60 Biopolis Street #02-01

department: Stem Cell

country: Singapore

institute: Genome Institute of Singapore

description: Gene expression data from mouse ES cells

hyb_protocol: 1.5 ug of cRNA were hybridized to Illumina Mouse Ref-6 V1 for 20 hr at 58oC on Ilumina Hybridization Oven.

scan_protocol: Illumina Mouse Ref-6 V1 was washed and stained with streptavidin-Cy3 before scanning using the Illumina Bead Reader.

data_processing: The data were analyzed using Bead Array suite software. Rank invariant normalization method was chosen to obtain the signal intensity.

source_name: R1 mouse embryonic stem (ES) cells

label_protocol: Biotinylated cRNA were prepared from 500 ng of total RNA according to the standard protocol (TotalPrep RNA Amplification Kit, Instruction Manual, Catalog #IL1791, Illumina).

growth_protocol: Mouse ES cells were cultured at 37C with 5% CO2. Mouse ES cells were maintained on mitomycin C-treated MEF feeders in Dulbecco's modified Eagle medium (DMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum (FBS; GIBCO), 0.055 mM Beta-mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF (Chemicon).

extract_protocol: Total RNA was extracted using Trizol (Invitrogen) and purified with RNeasy minikit (Qiagen).

value: Eset depletion

value: R1 mouse embryonic stem cells


title: NSCB_CGH_390K_16250602

description: This CGH experiment is designed to detect deletion, insertation, amplication, etc. in the sample relative to the reference.


title: Eset shRNA ES cells, biological replicate 2


title: NSCB_CGH_390K_16250102


title: Control shRNA ES cells, biological replicate 3

source_name: E14 mouse embryonic stem (ES) cells

growth_protocol: Control shRNA knocking down mouse ES cells were cultured at 37C with 5% CO2. Mouse ES cells were maintained on mitomycin C-treated MEF feeders in Dulbecco's modified Eagle medium (DMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum (FBS; GIBCO), 0.055 mM Beta-mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF (Chemicon) with 0.8ug/ml puromycin.

value: Control

value: E14 mouse embryonic stem cells


title: Eset shRNA ES cells, biological replicate 3


title: NSCB_CGH_390K_ES03_p92

value: human embryonic stem (hES)

extract_protocol:
1. Determine When to Harvest hES cells for DNA Extraction
A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity
required.
B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield.
C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells). One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage.
2. Trypsinize the Cells
A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment.
B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate.
C) Return the plate to the 37C incubator for 2-3 minutes.
D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope. Proceed to next step when colony edges begin to lift off the plate.
E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps.
F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution.
G) Gently pipet the solution up and down to mix thoroughly.
H) Centrifuge the tube for 5 minutes at 1000 RPM.
3. RNA Digestion
A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended.
B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing.
C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes.
4. Protein Digestion
A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by
vortexing. It is important that the solution is immediately and thoroughly mixed by
vortexing and pipetting to yield a homogenous solution.
NOTE: Do not add proteinase K directly to buffer AL.
B) Incubate the cell solution in a 70°C waterbath for 10 minutes.
5. DNA Precipitation and Separation of Cellular Components
A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing.
B) A white precipitate may form on addition of ethanol. This precipitate does not interfere with the DNeasy procedure.
C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 minute. Discard flowthrough and collection tube.
NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload
the spin column. The maximum number of hES cells that should be used in a DNA
extraction is 5x106 (normally, 2 wells of a 6-well plate). For larger amounts of starting material use multiple spin columns.
6. Wash the DNA
A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1.
B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube.
C)Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2.
D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube.
NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since
residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.
E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube.
7. Elution
A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane. Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute.
NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield.
B) Repeat elution step as described in 7 A).
NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step.
NOTE: More than 200μl should not be eluted into a 1.5 ml microcentrifuge


title: NSCB_CGH_390K_UC01_p07

description: NimbleGen scan protocol


title: Control shRNA ES cells, biological replicate 2

treatment_protocol: E14 mouse ES cells were transfected with control shRNA by lipofectamine.


title: NSCB_CGH_390K_16250202


title: Control shRNA ES cells, biological replicate 1


title: NSCB_CGH_390K_H9_p41


title: NSCB_CGH_390K_16236502


title: NSCB_CGH_390K_16250302


title: HEK293_MIF siRNA 2#_24h_Illumina

status: Public on Nov 03 2009

city: Shanghai

first: Lingfeng

last: Liu

address: Handan Road 220#

department: Life Science

country: China

institute: Fudan University

description: MIF siRNA(50pmol/ml)-mediated MIF knockdown in HEK293 cells

hyb_protocol: Refer to the Illumina Gene Expression System Manual

data_processing: Cubic spline normalization using Illumina BeadStudio Version "BSGX Version3.2.3"

label: Streptavidin Cy3

source_name: HEK293 cell, MIF Knockdown

label_protocol: As described in the illumina Human-6 chip processing protocol. Briefly, generation of biotin labelled cRNA from the product of RT cDNA synthesis and 2nd strand cDNA synthesis.

extract_protocol: Illumina TotalPrep RNA amplification kit (Ambion Catalog#IL1791)

value: HEK293 human embryonic kidney cell line

value: MIF depletion, siRNA-mediated

tag: siRNA concentration = 50pmol/ml


title: NSCB_CGH_390K_H9_p47_1


title: HEK293_Normal_24h_Illumina

description: The untreated HEK293 cells

scan_protocol: Refer to the Illumina Gene Expression System Manual

source_name: Normal HEK293 cell

value: control (untreated)


title: HEK293_Control RNA_24h_Illumina

description: The HEK293 cells treated with control RNA for 24h

source_name: HEK293, Control RNA treatment

value: control (control RNA)


title: NSCB_CGH_390K_16246002


title: NSCB_CGH_390K_16256902


title: NPC25

city: Durham

first: Wenbin

last: Wei

address: Stockton Road

department: Biosciences

country: United Kingdom

institute: Durham University

description: Stage IVA, T4, N2, M0, G4

hyb_protocol: Affymetrix Standard protocol

scan_protocol: Affymetrix Standard protocol

data_processing: The scanned images of microarray chips were analyzed using the Affymetrix GenChip Operating Software (GCOS) with the default settings except that the target signal was set to 100.

source_name: biopsy of undifferentiated NPC

label_protocol: The cDNA was synthesized, in vitro transcribed and labeled using standard Affymetrix protocols.

extract_protocol: RNA was extracted from biopsy samples using TRIzol Reagent (Invitrogen, USA) and purified by the RNeasy Mini Kit (Qiagen, UK).

value: Nasopharyngeal carcinoma

tag: stage


title: NSCB_CGH_390K_16236602


title: NPC24

description: Stage IVB, T2, N3, M0, G4


title: NPC23

description: Stage IVA, T4, N2, M0, G4


title: NPC22

description: Stage III, T1, N2, M0, G4


title: NPC20

description: Stage IIB, T2, N1, M0, G4


title: NPC15

description: Stage III, T2, N2, M0, G4


title: NPC21

description: Stage I, T1, N0, M0, G4


title: NPC11

description: Stage IVB, T4, N3, M0, G4


title: NPC14

description: Stage III, T2, N2, M0, G4


title: NPC12

description: Stage IIB, T1, N0, M0, G4


title: NPC13

description: Stage III, T1, N2, M0, G4


title: NPC18

description: Stage IVB, T3, N2, M0, G4


title: NPC9

description: Stage IIB, T2, N1, M0, G4


title: NPC8

description: Stage III, T2, N2, M0, G4


title: NPC5

description: Stage II, T2, N0, M0, G4


title: NPC3

description: Stage III, T2, N2, M0, G4


title: NPC6

description: Stage III, T3, N2, M0, G4


title: NPC7

description: Stage IVB, T3, N3, , G4

value: IVB, T3, N3, G4


title: NPC19

description: Stage IVA, T4, N2b, M0, G4

value: IVA, T4, N2b, M0, G4


title: NPC2

description: Stage IVC, T4, N3, M1, G4


title: Normal control 2

description: Non-malignant control

source_name: biopsy of non-malignant nasopharyngeal control

value: Normal


title: Toxoplasma gondii type I RH-JSR tachyzoite expression - sample 3

status: Public on Oct 26 2009

city: St Louis

first: Michael

middle: Sean

last: Behnke

state: MO

address: 660 S. Euclid Ave

department: Molecular Microbiology

institute: Washington University School of Medicine

description: type I RH-JSR tachyzoite expression using the Affymetrix Toxoplasma gondii Genechip

hyb_protocol: Standard Affymetrix protocol with 5ug labeled cRNA.

scan_protocol: Standard Affymetrix protocol

data_processing: GeneSpring 7.2 RMA, Data transformation: Set measurements less than 0.01 to 0.01, Per Chip and Per Gene: Median Polishing

organism: Toxoplasma gondii

source_name: Type I RH-JSR tachyzoite

label_protocol: Affymetrix One Cycle: 3ug starting RNA - Standard One Cycle protocol

growth_protocol: grown in human foreskin fibroblasts

extract_protocol: Parasites were purified from host cells and RNA was extracted using the Qiagen RNeasy kit (b-mercaptoethanol and DNase I treatment).

value: type I strain RH-JSR

tag: developmental stage

value: tachyzoite


title: Toxoplasma gondii type I RH-JSR tachyzoite expression - sample 2


title: NPC10

description: Stage IVB, T3, N3, M0, G4


title: NPC1

description: Stage III, T2, N2, M0, G4


title: Normal control 1

description: Non-malignant control


title: Toxoplasma gondii type I RH-JSR tachyzoite expression - sample 1


title: NPC4

description: Stage IIB, T1, N1, M0, G4


title: Toxoplasma gondii type I RH-ERP2009 tachyzoite expression - sample 2

description: type I RH-ERP2009 tachyzoite expression using the Affymetrix Toxoplasma gondii Genechip

source_name: Type I RH-ERP2009 tachyzoite

value: type I strain RH-ERP2009


title: Toxoplasma gondii type I RH-ERP2009 tachyzoite expression - sample 3


title: Normal control 3

description: Non-malignant control


title: Toxoplasma gondii type I RH-ERP2009 tachyzoite expression - sample 1


title: Toxoplasma gondii type I GT1 tachyzoite expression - sample 3

description: type I GT1 tachyzoite expression using the Affymetrix Toxoplasma gondii Genechip

source_name: Type I GT1 tachyzoite

value: type I strain GT1


title: Toxoplasma gondii type I GT1 tachyzoite expression - sample 2


title: Toxoplasma gondii type I GT1 tachyzoite expression - sample 1


title: blood_responder_patient36

status: Public on Oct 02 2009

city: Barcelona

first: Antonio

last: Julià

state: Barcelona

address: c/ Baldiri Reixac nº15

department: Rheumatology Research Group

country: Spain

institute: Vall d'Hebron Hospital Research Institute

description: Whole blood RNA, Infliximab responder

hyb_protocol: Biotin labeled cRNA (1.5ug) was hybridized to Sentrix(r) whole genome bead chips 6 x 2 (Illumina, San Diego, CA, USA)

scan_protocol: Scanning was performed on the Illumina(r) BeadStation 500x.

data_processing: Log2 transformation of BeadStudio raw data, gene filtering of Beadchip version2 RefSeq valid probes, quantile normalization using R.

source_name: Rheumatoid arthritis patient, whole blood, at 8:30-9:30 morning

label_protocol: Biotin labeled cRNA preparation for the Illumina platform was performed using the Ambion(r) Illumina RNA amplification kit (Ambion Europe, Huntington, Cambridgeshire, UK).

extract_protocol: Whole blood RNA was extracted from whole blood preserved in Paxgene tubes using the PAXgene RNA Isolation kit (Qiagen)

treatment_protocol: Paxgene tube conservation at -80ºC

value: responder

value: female

value: whole blood

tag: treatment stage

value: baseline (prior infliximab infusion)


title: blood_responder_patient34

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient31

description: Whole blood RNA, Infliximab responder

value: male


title: blood_responder_patient30

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient33

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient35

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient37

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient29

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient18

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient21

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient26

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient23

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient27

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient32

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient19

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient25

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient24

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient22

description: Whole blood RNA, Infliximab responder


title: HEK293_MIF siRNA 1#_24h_Illumina

description: MIF siRNA-mediated MIF knockdown in HEK293 cells

source_name: HEK293 cell, siRNA-mediated MIF knockdown

value: HEK293 human embryonic kidney cell line

tag: siRNA concentration = 100pmol/ml


title: blood_responder_patient20

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient28

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient16

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient15

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient14

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient13

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient12

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient17

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient8

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient10

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient11

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient9

description: Whole blood RNA, Infliximab responder


title: blood_nonresponder_patient6

description: Whole blood RNA, Infliximab non-responder

value: nonresponder


title: blood_nonresponder_patient3


title: blood_nonresponder_patient7


title: blood_responder_patient6

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient1

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient5

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient7

description: Whole blood RNA, Infliximab responder


title: blood_nonresponder_patient2


title: blood_responder_patient2

description: Whole blood RNA, Infliximab responder


title: blood_responder_patient4

description: Whole blood RNA, Infliximab responder


title: blood_nonresponder_patient5


title: blood_nonresponder_patient4


title: blood_nonresponder_patient1


title: blood_responder_patient3

description: Whole blood RNA, Infliximab responder


title: NSCB_CGH_385K_239_WA09

status: Public on Oct 01 2009

data_processing:
1. Image extraction: NimbleScan provided by NimbleGen is used to extract signal data from image files (*.TFF). Signals from the scan for Cy3 (green channel) are saved in a file named as chipID_532.pair while signals from the scan for Cy5 (red channel) are saved in a file named as chipID_635.pair.
2. Normalization: Signals from Cy3/Cy5 channels are transformed by logarithms to base 2 and normalized with loess or qspline (Workman et al., 2002) algorithms by R to resolve the systematic errors and bias introduced by the microarray experimental platform. The log ratios of the signals are obtained by subtracting the log transformed signals from one channel by the corresponding ones from another channel.
3. Data smoothing and break-points identification: Ratios of the normalized data are plotted by the corresponding genome locations. The data are smoothed by an adaptive weights smoothing algorithm (GLAD R-package, Wang et al., 2005), break-points are identified by a binary segmentation algorithm (DNAcopy R-package, Olshen et al., 2004) or by the segmentation algorithm available within Nimbelgen’s Nimblescan software.
4. Data visualization: CGH results can be saved as SignalMap GFF files and can be visualized by SignalMap provided by NimbleGen. CGH results can also be visualized by other software, such as aCGHViewer (Shankar et al., 2006), CHARMView (Myers et al., 2005), etc. However, different software requires different input format.

source_name: WA09 DNA380

extract_protocol:
1. Determine When to Harvest hES cells for DNA Extraction
A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity required.
B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield.
C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells). One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage.
2. Trypsinize the Cells
A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment.
B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate.
C) Return the plate to the 37C incubator for 2-3 minutes.
D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope. Proceed to next step when colony edges begin to lift off the plate.
E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps.
F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution.
G) Gently pipet the solution up and down to mix thoroughly.
H) Centrifuge the tube for 5 minutes at 1000 RPM.
3. RNA Digestion
A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended.
B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing.
C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes.
4. Protein Digestion
A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by vortexing. It is important that the solution is immediately and thoroughly mixed by vortexing and pipetting to yield a homogenous solution.
NOTE: Do not add proteinase K directly to buffer AL.
B) Incubate the cell solution in a 70°C waterbath for 10 minutes.
5. DNA Precipitation and Separation of Cellular Components
A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing.
B) A white precipitate may form on addition of ethanol. This precipitate does not interfere with the DNeasy procedure.
C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 minute. Discard flowthrough and collection tube.
NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload the spin column. The maximum number of hES cells that should be used in a DNA extraction is 5x106 (normally, 2 wells of a 6-well plate). For larger amounts of starting material use multiple spin columns.
6. Wash the DNA
A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1.
B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube.
C) Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2.
D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube.
NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.
E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube.
7. Elution
A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane. Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute.
NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield.
B) Repeat elution step as described in 7 A).
NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step.
NOTE: More than 200μl should not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy spin column will come into contact with the elute.

value: human embryonic stem (hES) cell line NSCB# 9016, CGH# 239

value: human embryonic stem (hES) cell line H9 [reference]


title: NSCB_CGH_385K_238_BG03

value: human embryonic stem (hES) cell line NSCB# 4012, CGH# 238


title: NSCB_CGH_385K_240_ES02

value: human embryonic stem (hES) cell line NSCB# 0695, CGH# 240


title: NSCB_CGH_385K_223_ES02

value: human embryonic stem (hES) cell line NSCB# 8026, CGH# 223


title: NSCB_CGH_385K_224_WA09

value: human embryonic stem (hES) cell line NSCB# 1676, CGH# 224


title: NSCB_CGH_385K_234_WA09

value: human embryonic stem (hES) cell line NSCB# 3048, CGH# 234


title: NSCB_CGH_385K_225_ES02

value: human embryonic stem (hES) cell line NSCB# 6475, CGH# 225


title: NSCB_CGH_385K_233_BG03

value: human embryonic stem (hES) cell line NSCB# 7428, CGH# 233


title: NSCB_CGH_385K_236_TE04

value: human embryonic stem (hES) cell line NSCB# 2793, CGH# 236


title: NSCB_CGH_385K_222_WA09

value: human embryonic stem (hES) cell line NSCB# 4361, CGH# 222


title: NSCB_CGH_385K_237_WA09

value: human embryonic stem (hES) cell line NSCB# 3992, CGH# 238


title: NSCB_CGH_390K_220_BG03

value: human embryonic stem (hES) cell line NSCB# 1304, CGH# 220


title: NSCB_CGH_385K_221_TE03

value: human embryonic stem (hES) cell line NSCB# 3683, CGH# 221


title: NSCB_CGH_385K_229_TE03

value: human embryonic stem (hES) cell line NSCB# 1878, CGH# 229


title: NSCB_CGH_385K_227_TE03

value: human embryonic stem (hES) cell line NSCB# 6416, CGH# 227


title: NSCB_CGH_385K_232_ES02

value: human embryonic stem (hES) cell line NSCB# 9866, CGH# 232


title: NSCB_CGH_385K_226_TE04

value: human embryonic stem (hES) cell line NSCB# 5301, CGH# 226


title: NSCB_CGH_385K_235_WA07

value: human embryonic stem (hES) cell line NSCB# 7594, CGH# 235

value: human embryonic stem (hES) cell line H9 [reference]


title: NSCB_CGH_385K_230_WA09

value: human embryonic stem (hES) cell line NSCB# 9854, CGH# 230


title: NSCB_CGH_390K_228_BG03

value: human embryonic stem (hES) cell line NSCB# 6257, CGH# 228


title: NSCB_CGH_390K_213_BG03

source_name: BG03 DNA300

value: human embryonic stem (hES) cell line NSCB# 6133, CGH# 213


title: NSCB_CGH_385K_219_WA09

value: human embryonic stem (hES) cell line NSCB# 0806, CGH# 219


title: NSCB_CGH_385K_231_TE03

source_name: TE03 DNA371

value: human embryonic stem (hES) cell line NSCB# 8530, CGH# 231


title: Brain_animal-4165_line-62

status: Public on Sep 22 2009

city: Tjele

first: Jakob

last: Hedegaard

address: PO-box 50

department: Department of Genetics and Biotechnology

country: Denmark

institute: Aarhus University, Faculty of Agricultural Sciences

description: Expression profiling of 179 chicken brains sampled from three lines - 60 chickens from a high feather pecking line (HP), 60 from a low feather pecking line (LP) and 59 from a random bred control line (C). Expression profiling was conducted by hybridising each sample together with a common reference, consisting of brain RNA from an unrelated F1 animal

hyb_protocol: The slides were hybridized in a Discovery XT hybridization station (Ventana Discovery Systems, Tucson, AZ, USA). Transfer Chip Prep-2 from 4 ºC to room temperature 1 hour before use. Prepare ChipSpread by mixing equal volumes of ChipSpread A (20 mg/mL BSA, 4x SSC, 0.5 mg/mL sodium azide) and B (formamide; 2 mg/mL SDS) and incubate at room temperature for 1 hour before use. A total of 2.5 mL is needed per slide. Print labels, trim them and place them on the slides. Mix the Chip Map reagents (Chip Prep-1, -2 and - 3) by inversion, remove the cap and place the reagents in the Discovery. Place the slides in the machine and initiate the run. Cover slide with 2.5 mL ChipSpread when the message appears (after few minutes). The machine now runs for app. 1.5 hours to pre-hybridize the slides. Heat a waterbath to 90°C or use a PCR machine. Mix the Chiphybe, add 200 µL to the sample (<20 µL) and mix carefully. Heat the sample mixture at 90°C for 3 minutes and mix carefully by pipetting. Press "button" on the machine which then prepares the slides for hybridization. When the message appears apply 100 µL Chiphybe onto each slide. Then apply the samples (200 µL) onto each slide, press "button" and the machine hybridizes at 45 ºC for 6 hours. Wipe oil from backside of slides using a clean-room napkin and place slides in the slide-holder from the High Throughput Wash Station (Telechem, cat.no. HTW) placed in a mTub filled with RiboWash. If processing more than 20 slides, place equal number of slides in two slide-holders and continue in parallel. Dip the slides 20 times in RiboWash. Transfer the slide-holder to a HTW filled with RiboWash and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with RiboWash and repeat the wash. Dip the slide-holder in 2x SSC filled in a mTub (200 mL 20x SSC, Elga H2O + 1800 mL water). Transfer the slide-holder to a HTW filled with 2x SSC and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with 2x SSC and repeat the wash. Dip the slide-holder 10 times in 0.1x SSC filled in a mTub (5 mL 20x SSC, Elga H2O + 995 mL water) and leave the holder submerged in 0.1x SSC. Transfer the slides to a mBox slide holder placed in a mTub filled with Elga H2O. Dry arrays by centrifugation (at 300 x g for 4 min placed in a mBox).

scan_protocol: Scanner: ScanArray Express HT system (version 3.0, Perkin Elmer), 5 µm resolution, 100 % laser power and PMT adjusted individually for each channel. Image analysis software: GenePix Pro (version 6.0.1.27, Molecular Devices) using FeatureType = Irregular Filled and MorphologicalClosingFollowedByOpening as background measurement.

data_processing: Statistical analysis was carried out in the R computing environment (version 2.5.0 for Windows) using the package Linear Models for Microarray Analysis (Limma, version 2.10.0) which is part of the Bioconductor project. The log2-transformed ratios of Alexa-647 to Alexa-555 (median intensities, not background corrected) were normalized within-slide using loess with default parameters as implemented in Limma. Further analysis: Single gene analysis: Limma version 2.10.0; Gene set enrichment analysis :  Limma version 2.10.0 (GO/KEGG) / topGO version 1.2.1; 2 D clustering: heatmap.2 option in gplots version 2.3.2; co-expression: coXpress version 1.3.

organism: Gallus gallus

label_protocol: Ten µg Total-RNA from each individual sample was labeled with Alexa-647 using the SuperScript Direct cDNA labeling System (Invitrogen). The labelled cDNA was purified using the NucleoSpin 96 Extract II PCR Clean-up kit (Macherey-Nagel).

growth_protocol: The birds were reared in floor pens covered with a 5-cm thick layer of wood shavings. The temperature was 34 C at 1-day-old and gradually reduced to 20 C at 8 weeks of age. This temperature was kept throughout the rest of the experiment. The light regime was 12L:12D from 0 to 14 weeks and then 1 hour light per week was added until the light regime was 16L:8D at 18 weeks of age. Thereafter the light regime was constant until the bird was used in the experiment.

extract_protocol: The whole brain was homogenized in liquid nitrogen using a TissueLyser (Qiagen/Retsch) fitted with 50 mL stainless steel grinding jars and 20 mm grinding balls. Total-RNA was purified and DNase treated using NucleoSpin RNA L (Macherey-Nagel) following the enclosed protocol.

treatment_protocol: From 1-day-old until 18 weeks of age: group housing. 18 weeks - 33 weeks of age: individual housing to collect the eggs. At 33 weeks of age the chickens were divided into groups of 20 hens for each line, separately. The birds had a week to get used to the new environment and group composition. At 34 weeks of age the chickens were subject to a social feather pecking test of 3h between 14:00h to 17:00h. This test is used in the selection procedure of the selection lines and is described in detail by Kjaer et al. [Kjaer, J.B., Sørensen, P, and G. Su. 2001. Divergent selection on feather pecking behaviour in laying hens (Gallus gallus domesticus). Appl. Anim. Behav. Sci. 71: 229-239]. The next day between 8:00h and 12:00h the bird was decapitated and the whole brain was taken and frozen directly in liquid nitrogen and stored at - 80 C until further use.

value: White Leghorn High Feather Pecking line (Gallus gallus domesticus); This is a line which is selected for an increase in feather pecking behaviour for 8 generations

value: female

value: 34 weeks

value: whole brain

tag: severe  feather pecking

tag: aggressive  feather pecking

tag: gentle  feather pecking

tag: receiving severe pecks

tag: receiving aggressive pecks

tag: receiving gentle pecks

tag: cage

label_protocol: Ten µg Total-RNA from the reference sample was labelled with Alexa-555 using the SuperScript Direct cDNA labeling System (Invitrogen). The labelled cDNA was purified using the NucleoSpin 96 Extract II PCR Clean-up kit (Macherey-Nagel). The labelled reference samples were mixed and divided into aliquots before combining it with the labelled sample.

treatment_protocol: From 1-day-old until 18 weeks of age: group housing. 18 weeks - 34 weeks of age: individual housing to collect the eggs. At 34 weeks of age: the bird was taken out of his cage and between 8:00h and 12:00h the bird was decapitated and the whole brain was taken and frozen directly in liquid nitrogen and stored at - 80 C until further use.

value: White Leghorn F1 animal from a cross between high feather pecking line x low feather pecking line (Gallus gallus domesticus)

value: whole brain.


title: NSCB_CGH_385K_211_TE03

tag: cell line

value: human embryonic stem (hES) cell line NSCB# 8246, CGH# 211


title: NSCB_CGH_385K_212_ES02

value: human embryonic stem (hES) cell line NSCB# 1609, CGH# 212


title: Brain_animal-4123_line-62

tag: strain

tag: gender

tag: age

tag: tissue


title: Brain_animal-4680_line-6

value: White Leghorn Control line (Gallus gallus domesticus); This is a random mating line maintained for 8 generations


title: Brain_animal-4846_line-6


title: Brain_animal-4790_line-6


title: NSCB_CGH_385K_207_ES03

value: human embryonic stem (hES) cell line NSCB# 2668, CGH# 207


title: NSCB_CGH_385K_214_TE03

value: human embryonic stem (hES) cell line NSCB# 7867, CGH# 214


title: NSCB_CGH_385K_209_WA09

value: human embryonic stem (hES) cell line NSCB# 2988, CGH# 209

value: human embryonic stem (hES) cell line H9 [reference]


title: NSCB_CGH_385K_210_WA09

value: human embryonic stem (hES) cell line NSCB# 5268, CGH# 210


title: Brain_animal-4382_line-61

value: White Leghorn Low Feather Pecking line (Gallus gallus domesticus); This is a line which is selected for a decrease in feather pecking behaviour for 8 generations


title: NSCB_CGH_385K_161_UC01

value: human embryonic stem (hES) cell line NSCB#6338 CGH#161


title: Brain_animal-4526_line-61


title: NSCB_CGH_385K_205_WA09

source_name: WA09 DNA155

value: human embryonic stem (hES) cell line NSCB# 9510, CGH# 205


title: Brain_animal-4748_line-6


title: NSCB_CGH_385K_204_UC06

value: human embryonic stem (hES) cell line NSCB# 4487, CGH# 204


title: NSCB_CGH_385K_186_ES04

value: human embryonic stem (hES) cell line NSCB# 0654B, CGH# 186


title: Brain_animal-4015_line-62

value: White Leghorn High Feather Pecking line (Gallus gallus domesticus); This is a line which is selected for an increase in feather pecking behaviour for 8 generations

value: female

value: 34 weeks


title: NSCB_CGH_385K_206_UC06

source_name: UC06 DNA159

value: human embryonic stem (hES) cell line NSCB# 1594, CGH# 206


title: Brain_animal-4443_line-61

value: whole brain

value: White Leghorn F1 animal from a cross between high feather pecking line x low feather pecking line (Gallus gallus domesticus)

value: whole brain.


title: NSCB_CGH_385K_198_WA09

description:
Specimen normal per pathology report.
Agilent BioAnalyzer 2100 RNA Integrity Number=10

value: human embryonic stem (hES) cell line NSCB# 7842, CGH# 198


title: NSCB_CGH_385K_200_ES03

value: human embryonic stem (hES) cell line NSCB# 8665B, CGH# 200


title: NSCB_CGH_385K_201_ES03

value: human embryonic stem (hES) cell line NSCB# 9180, CGH# 201


title: NSCB_CGH_385K_197_ES03

value: human embryonic stem (hES) cell line NSCB# 8268, CGH# 197

value: human embryonic stem (hES) cell line H9 [reference]


title: NSCB_CGH_385K_202_WA09

value: human embryonic stem (hES) cell line NSCB# 1133, CGH# 202


title: Brain_animal-4843_line-6

value: White Leghorn Control line (Gallus gallus domesticus); This is a random mating line maintained for 8 generations


title: Brain_animal-4655_line-6


title: Brain_animal-4506_line-61

value: White Leghorn Low Feather Pecking line (Gallus gallus domesticus); This is a line which is selected for a decrease in feather pecking behaviour for 8 generations


title: NSCB_CGH_385K_187_WA14

value: human embryonic stem (hES) cell line NSCB# 8808B, CGH# 187


title: Brain_animal-4769_line-6


title: Brain_animal-4356_line-61


title: Brain_animal-4420_line-61


title: Brain_animal-4691_line-6


title: Brain_animal-4765_line-6


title: Brain_animal-4760_line-6


title: Brain_animal-4153_line-62


title: Brain_animal-4093_line-62


title: NSCB_CGH_385K_185_UC01

value: human embryonic stem (hES) cell line NSCB# 2538, CGH# 185


title: Brain_animal-4737_line-6


title: Brain_animal-4358_line-61


title: Brain_animal-4620_line-6


title: Brain_animal-4116_line-62

value: White Leghorn High Feather Pecking line (Gallus gallus domesticus); This is a line which is selected for an increase in feather pecking behaviour for 8 generations


title: Brain_animal-4285_line-62

value: female


title: Brain_animal-4493_line-61

value: 34 weeks


title: Brain_animal-4771_line-6


title: Brain_animal-4351_line-61


title: Brain_animal-4779_line-6


title: Brain_animal-4653_line-6


title: Brain_animal-4617_line-6


title: Brain_animal-4344_line-61


title: Brain_animal-4741_line-6


title: Brain_animal-4767_line-6


title: Brain_animal-4753_line-6


title: Brain_animal-4252_line-62


title: Brain_animal-4566_line-61


title: Brain_animal-4371_line-61


title: Brain_animal-4870_line-6


title: Brain_animal-4126_line-62


title: Brain_animal-4792_line-6


title: Brain_animal-4379_line-61


title: Brain_animal-4646_line-6


title: Brain_animal-4401_line-61


title: Brain_animal-4836_line-6


title: Brain_animal-4658_line-6


title: Brain_animal-4056_line-62


title: Brain_animal-4337_line-61


title: Brain_animal-4745_line-6


title: Brain_animal-4342_line-61


title: Brain_animal-4331_line-61


title: Brain_animal-4750_line-6


title: Brain_animal-4248_line-62


title: Brain_animal-4777_line-6


title: Brain_animal-4650_line-6


title: Brain_animal-4346_line-61


title: Brain_animal-4796_line-6


title: Brain_animal-4839_line-6


title: Brain_animal-4725_line-6


title: Brain_animal-4360_line-61


title: Brain_animal-4228_line-62


title: Brain_animal-4283_line-62


title: Brain_animal-4477_line-61


title: Brain_animal-4433_line-61


title: Brain_animal-4307_line-62


title: Brain_animal-4637_line-6


title: Brain_animal-4720_line-6


title: Brain_animal-4238_line-62


title: Brain_animal-4735_line-6


title: Brain_animal-4410_line-61


title: Brain_animal-4395_line-61


title: Brain_animal-4246_line-62


title: Brain_animal-4028_line-62


title: Brain_animal-4042_line-62


title: Brain_animal-4709_line-6


title: Brain_animal-4729_line-6


title: Brain_animal-4697_line-6


title: Brain_animal-4147_line-62


title: Brain_animal-4413_line-61


title: Brain_animal-4292_line-62


title: Brain_animal-4832_line-6


title: Brain_animal-4685_line-6


title: Brain_animal-4053_line-62