title: NSCB_expression_380K_ES03_2

status: Public on May 20 2008

type: mixed

city: Madison

first: Melodee

last: Patterson

state: WI

address: 510 Charmany Dr Ste 58 & 59

institute: WiCell

description: Gene expression chip experiments are used to detect the expression level of hESC and differentiation markers in hESC cell lines.

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

scan_protocol: 1. Turn on the scanner and allow it to warm up for at least 15 minutes before scanning. 2. Open the Gene Pix Scan 6.0 program. 3. Place the chip in the scanner, with the label facing down and outward. 4. Open hardware settings and make sure the pixel size is 5 and the power is 100% for both channels. 5. Do a Preview Scan to adjust the scan frame for the array. Use levels 750 for the 635 (Cy5) channel and 550 for the 532 (Cy3) channel. 6. Perform Data Scans until the PMT levels for the 635 and 532 channels have been adjusted. 7. Adjust the PMT level of the 635 channel so the histogram is leveling off at 1e-5. 8. Adjust the PMT level of the 532 channel so the median (found by the rectangular region measuring tool) is equal to or no more than 30 points higher than the median of the 635 channel. 9. Save image as Single-image TIFF File (*.tif) and make sure both channels are checked. The image files are named chipID_635.tif and chipID_532.tif and stored on the WiCell Iceland server under the scan date (yyyymmdd). 10. The image files are ready for analysis with the NimbleScan software.

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:
Signal intensities from RNA and gDNA samples are normalized with Robust Multiple-chip Analysis (RMA) algorithm (Irizarry et al., 2003) separately. A median-adjusted ratio is calculated for each gene by dividing its signal intensity from the RNA sample by the sum of the corresponding signal intensity from the gDNA sample and median signal intensity of all genes from the gDNA channel.

label_protocol:
1. Dry the sample in the Speed Vac set at 30°C.
2. Resuspend the sample in 4.5μl 0.1M Na2CO3, pH 9.0.
3. Resuspend the Cy5 NHS ester in 4.5μl DMSO
4. Add Cy5 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:
6.1. Add 35μl 100mM NaOAc, pH 5.2 to the sample.
6.2. Add 250μl PB buffer to the sample and load the sample on the column.
6.3. Spin at 13.000 rpm for 1 minute and remove the flow through.
6.4. Add 750μl PE buffer to the column.
6.5. Spin at 13.000 rpm for 1 minute and remove the flow through.
6.6. Spin at 13.000 rpm for 1 minute.
6.7. Transfer the column to new tube.
6.8. Add 30μl EB buffer and incubate for 1 minute on the column.
6.9. Elute once by centrifuging at 13.000 rpm for 1 minute.

extract_protocol:
1. In a hood equipped with an aspirator, remove spent media from the wells of hES cells.
2. Wash each well once with 1ml of PBS. Aspirate the PBS.
3. In the fume hood, add 1 ml of Trizol per well using a 1ml pipetman. Use the pipet tip to scrape the cells into solution and make sure no cells are left behind.
4. Transfer the cells from each well into a separate 1.5ml eppendorf tube.
5. Add 200μl of chloroform into each tube and mix by pipetting up and down. Use a 1ml Pipetman.
6. Spin the tubes at 16000 RCF for 20 minutes at 4°C.
7. Transfer the upper 500μl of the aqueous phase into a new 1.5ml eppendorf tube.
8. Add 500μl of isopropanol to the new tube, and it mix by pipetting up and down.
9. Let the samples sit at room temperature for 15 minutes.
10. Pellet the RNA by spining the tubes at 16000 RCF for 20 minutes at 4°C.
11. Wash the pelleted RNA using 70% ethanol. Slowly add 1ml of 70% ethanol to the RNA, cap the tube, and wash the pellet by flipping the tube twice. If the pellet is dislodged, perform a quick spin and then carefully decant the ethanol.
12. Dry the RNA in a vacuum; however, take care not to over dry it as that makes RNA resuspension difficult.
13. Resuspend the RNA in 25μl of nuclease free water.
14. Use a nanodrop spectrophotometer to measure the concentration and purity of RNA (A260/A280). Record these values for the gene expression submission form.

value: human embryonic stem cell (hESC)

tag: cell line

value: ES03 human embryonic stem cell mRNA

source_name: hESC H1 DNA

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.

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.


title: NSCB_expression_380K_HSF1.14

country: USA

molecule: total RNA

organism: Homo sapiens

tag: cell type

value: HSF1.14 human embryonic stem cell mRNA

molecule: genomic DNA


title: NSCB_expression_380K_H9_2

value: H9 human embryonic stem cell mRNA


title: NSCB_expression_380K_H9_1


title: Nuclear scaffold and matrix attachment of HeLa chromatin (NaCl_2)

status: Public on Feb 27 2009

type: genomic

city: Detroit

first: Stephen

middle: A

last: Krawetz

state: MI

address: 275 East Hancock, CS Mott Center

institute: Wayne State University

description: The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in HeLa cells.

hyb_protocol: Hybridized at NimbleGen facilities in accordance with NimbleGen protocol

scan_protocol: Scanned at NimbleGen facilities in accordance with NimbleGen protocol

data_processing: Dual channel array data was q spline normalized using the NimbleGen data analysis suite. Normalized signal ratios were analyzed for enrichment in either the loop or matrix fraction.

source_name: Scaffold attachment profile of HeLa DNA: NaCl Loop Replicate 2

label_protocol: Labeled at NimbleGen facilities in accordance with manufacturer's protocol

extract_protocol: Nuclear halos were prepared in solution from isolated HeLa nuclei with exposure to 2 M NaCl using approximately 1 x 107 cells. After extraction, the halos were pelleted at 1000 x g for 5 minutes at 4°C then washed gently in REact® 3 restriction buffer (Invitrogen, Carlsbad, CA) on a rocker platform for 20 minutes at room temperature then centrifuged at 1000 x g for 5 minutes at 4°C then the supernatant discarded. This washing procedure was repeated an additional two times. After the third wash, the halos were resuspended in restriction buffer and the loops separated from the nuclear matrix associated DNA by digestion with 400 U of EcoRI (Invitrogen, Carlsbad, CA) at 37°C for 3 hours. Subsequent to restriction digestion, the matrix fraction was pelleted by centrifugation at 16,000 x g for 5 minutes at 4°C and the loop containing supernatant was removed and placed in a separate tube. The matrix fraction was resuspended, and then washed in restriction buffer, immediately pelleted at 16,000 x g for 5 minutes at 4°C and supernatant discarded. The nuclear matrix containing pellet fraction was washed an additional two times. Both loop and matrix restriction fragments were then freed from any nuclear proteins by overnight digestion at 55°C with 50 µg/mL of proteinase K buffered with 50 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 25 mM EDTA and 0.5% SDS. DNA was recovered and purified using a Quantum-prep matrix (BioRad, Hercules, CA) then resuspended in deionized water.

tag: gender

value: female

value: cultured HeLa S3 cells

source_name: Scaffold attachment profile of HeLa DNA: NaCl Matrix Replicate 2


title: Nuclear scaffold attachment of Aortic Adventitial Fibroblast (AoAF) chromatin Replicate 1

status: Public on Mar 11 2009

address: 275 East Hancock Avenue

description: The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications.  Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively.  We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in AoAF cells.

hyb_protocol: Hybridized at NimbleGen facilities as per NimbleGen protocol

scan_protocol: Scanned at NimbleGen facilities as per NimbleGen protocol

data_processing: Dual channel array data was q spline normalized using the NimbleGen data analysis suite.  Normalized signal ratios were analyzed for enrichment in either the loop or matrix fraction.

source_name: Scaffold attachment profile of AoAF DNA: LIS Loop Replicate 1

label_protocol: Labeled at NimbleGen facilities

extract_protocol: Nuclear halos were prepared in solution from isolated AoAF nuclei with dounce homogenization in the presence of 25 mM LIS (lithium 3,5-diiodosalicylate) using approximately 1 x 107 cells.  After extraction, the halos were pelleted at 1000 x g for 5 minutes at 4°C then washed gently in REact® 3 restriction buffer (Invitrogen, Carlsbad, CA) on a rocker platform for 20 minutes at room temperature then centrifuged at 1000 x g for 5 minutes at 4°C then the supernatant discarded.  This washing procedure was repeated an additional two times.  After the third wash, the halos were resuspended in restriction buffer and the loops separated from the nuclear scaffold associated DNA by digestion with 400 U of EcoRI (Invitrogen, Carlsbad, CA) at 37°C for 3 hours.  Subsequent to restriction digestion, the scaffold fraction was pelleted by centrifugation at 16,000 x g for 5 minutes at 4°C and the loop containing supernatant was removed and placed in a separate tube.  The scaffold fraction was resuspended, and then washed in restriction buffer, immediately pelleted at 16,000 x g for 5 minutes at 4°C and supernatant discarded.  The nuclear scaffold containing pellet fraction was washed an additional two times.  Both loop and scaffold restriction fragments were then freed from any nuclear proteins by overnight digestion at 55°C with 50 µg/mL of proteinase K buffered with 50 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 25 mM EDTA and 0.5% SDS.  DNA was recovered and purified using a Quantum-prep matrix (BioRad, Hercules, CA) then resuspended in deionized water.

value: Aortic adventitial fibroblast cell line

source_name: Scaffold attachment profile of AoAF DNA: LIS Scaffold Replicate 1


title: NSCB_expression_380K_H13C

value: H13C human embryonic stem cell mRNA


title: Nuclear scaffold and matrix attachment of HeLa chromatin (NaCl_1)

description: The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications.  Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively.  We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in HeLa cells.

source_name: Scaffold attachment profile of HeLa DNA: NaCl Loop Replicate 1

extract_protocol: Nuclear halos were prepared in solution from isolated HeLa nuclei with exposure to 2 M NaCl using approximately 1 x 107 cells.  After extraction, the halos were pelleted at 1000 x g for 5 minutes at 4°C then washed gently in REact® 3 restriction buffer (Invitrogen, Carlsbad, CA) on a rocker platform for 20 minutes at room temperature then centrifuged at 1000 x g for 5 minutes at 4°C then the supernatant discarded.  This washing procedure was repeated an additional two times.  After the third wash, the halos were resuspended in restriction buffer and the loops separated from the nuclear matrix associated DNA by digestion with 400 U of EcoRI (Invitrogen, Carlsbad, CA) at 37°C for 3 hours.  Subsequent to restriction digestion, the matrix fraction was pelleted by centrifugation at 16,000 x g for 5 minutes at 4°C and the loop containing supernatant was removed and placed in a separate tube.  The matrix fraction was resuspended, and then washed in restriction buffer, immediately pelleted at 16,000 x g for 5 minutes at 4°C and supernatant discarded.  The nuclear matrix containing pellet fraction was washed an additional two times.  Both loop and matrix restriction fragments were then freed from any nuclear proteins by overnight digestion at 55°C with 50 µg/mL of proteinase K buffered with 50 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 25 mM EDTA and 0.5% SDS.  DNA was recovered and purified using a Quantum-prep matrix (BioRad, Hercules, CA) then resuspended in deionized water.

source_name: Scaffold attachment profile of HeLa DNA: NaCl Matrix Replicate 1


title: Nuclear scaffold and matrix attachment of HeLa chromatin (LIS_2)

source_name: Scaffold attachment profile of HeLa DNA: LIS Loop Replicate 2

extract_protocol: Nuclear halos were prepared in solution from isolated HeLa nuclei with dounce homogenization in the presence of 25 mM LIS using approximately 1 x 107 cells.  After extraction, the halos were pelleted at 1000 x g for 5 minutes at 4°C then washed gently in REact® 3 restriction buffer (Invitrogen, Carlsbad, CA) on a rocker platform for 20 minutes at room temperature then centrifuged at 1000 x g for 5 minutes at 4°C then the supernatant discarded.  This washing procedure was repeated an additional two times.  After the third wash, the halos were resuspended in restriction buffer and the loops separated from the nuclear scaffold associated DNA by digestion with 400 U of EcoRI (Invitrogen, Carlsbad, CA) at 37°C for 3 hours.  Subsequent to restriction digestion, the scaffold fraction was pelleted by centrifugation at 16,000 x g for 5 minutes at 4°C and the loop containing supernatant was removed and placed in a separate tube.  The scaffold fraction was resuspended, and then washed in restriction buffer, immediately pelleted at 16,000 x g for 5 minutes at 4°C and supernatant discarded.  The nuclear scaffold containing pellet fraction was washed an additional two times.  Both loop and scaffold restriction fragments were then freed from any nuclear proteins by overnight digestion at 55°C with 50 µg/mL of proteinase K buffered with 50 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 25 mM EDTA and 0.5% SDS.  DNA was recovered and purified using a Quantum-prep matrix (BioRad, Hercules, CA) then resuspended in deionized water.

source_name: Scaffold attachment profile of HeLa DNA: LIS Matrix Replicate 2


title: Nuclear matrix attachment of Aortic Adventitial Fibroblast (AoAF) chromatin Replicate 2

source_name: Matrix attachment profile of AoAF DNA: NaCl Loop Replicate 2

extract_protocol: Nuclear halos were prepared in solution from isolated AoAF nuclei with exposure to 2 M NaCl using approximately 1 x 107 cells.  After extraction, the halos were pelleted at 1000 x g for 5 minutes at 4°C then washed gently in REact® 3 restriction buffer (Invitrogen, Carlsbad, CA) on a rocker platform for 20 minutes at room temperature then centrifuged at 1000 x g for 5 minutes at 4°C then the supernatant discarded.  This washing procedure was repeated an additional two times.  After the third wash, the halos were resuspended in restriction buffer and the loops separated from the nuclear matrix associated DNA by digestion with 400 U of EcoRI (Invitrogen, Carlsbad, CA) at 37°C for 3 hours.  Subsequent to restriction digestion, the matrix fraction was pelleted by centrifugation at 16,000 x g for 5 minutes at 4°C and the loop containing supernatant was removed and placed in a separate tube.  The matrix fraction was resuspended, and then washed in restriction buffer, immediately pelleted at 16,000 x g for 5 minutes at 4°C and supernatant discarded.  The nuclear matrix containing pellet fraction was washed an additional two times.  Both loop and matrix restriction fragments were then freed from any nuclear proteins by overnight digestion at 55°C with 50 µg/mL of proteinase K buffered with 50 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 25 mM EDTA and 0.5% SDS.  DNA was recovered and purified using a Quantum-prep matrix (BioRad, Hercules, CA) then resuspended in deionized water.

source_name: Matrix attachment profile of AoAF DNA: NaCl Matrix Replicate 2


title: Nuclear matrix attachment of Aortic Adventitial Fibroblast (AoAF) chromatin Replicate 1

source_name: Matrix attachment profile of AoAF DNA: NaCl Loop Replicate 1

source_name: Matrix attachment profile of AoAF DNA: NaCl Matrix Replicate 1


title: NSCB_expression_380K_ES06

value: ES06 human embryonic stem cell mRNA


title: NSCB_expression_380K_WA09_35

value: WA09_35 human embryonic stem cell mRNA


title: NSCB_expression_380K_HSF1_1

value: HSF1 human embryonic stem cell mRNA


title: NSCB_expression_380K_ES04

value: ES04 human embryonic stem cell mRNA


title: NHGRI_WM1791C

status: Public on Jan 24 2001

type: RNA

city: Bethesda

first: Michael

last: Bittner

state: MD

department: Cancer Genetics Branch

institute: NHGRI, NIH

description:
Biopsy location: unknown
Patient sex: unknown
Patient age: unknown
Passage number: 52
p16 mutation status: ND
Invasive ability: 4.6 +/- 0.3%
Vasculogenic mimicry: +
Gel contraction: ND
Cell motility: 141.00 +/- 11.40
Scratch wound (%): 71

See http://www.nhgri.nih.gov/DIR/Microarray/Nature_paper.pdf
and http://www.nhgri.nih.gov/DIR/Microarray/Melanoma_Supplement/index.html

data_processing: Probe-level signal intensities were normalized to a common reference array using quantile normalization and combined to form SNP-level signal intensities using the dChip model-based expression (PM/MM) method . Genome-wide copy number estimates were obtained using tangent normalization, in which tumor signal intensities are divided by signal intensities from the linear combination of all normal samples that is most similar to the tumor (as described in Beroukhim et al, In Press; and Getz et al., In Preparation). Cancer samples were normalized against a pool of 1,480 reference samples derived from multiple studies; the code and reference data necessary to replicate this analysis are available for download in GEO. Genotype Call (SNP call) are provided in .CHP.TXT files: AA, AB, BB, NC, and NoCall.

source_name: melanoma


title: 19 (R) Hsab-03-46

status: Public on Oct 24 2005

first: Natalia

last: Shulzhenko

address: 9000 Rockville Pike, Building 4, room 111

department: NIAID

institute: NIH

description:
aRNA:19
Patient&sample ID:	D119
Sex:	female
Basic disease:	Chagas cardiomyopathy
Days after Tx:	119
Histology (ISHLT grade):	1B
Clinical history notes:	2 months after starting treatment for Chagas
Final Clinical diagnosis:	R
Symbols and abbreviations: Patient&Sample ID: letter=patient ID,  number=days after Tx. Tx=transplantation; ISHLT=International Society for heart and Lung Transplantation; R=rejection; Ch=Chagas disease reactivation; N=non-rejection&non-Chagas.
Keywords = cardiac transplant
Keywords = acute rejection
Keywords = infection
Lot batch = Hsab-03

source_name: reference pool, Cy5

source_name: endomyocardial biopsy, Cy3


title: NSCB_expression_380K_ES03_1


title: NSCB_expression_380K_0583

value: Unknown human embryonic stem cell mRNA


title: NSCB_expression_380K_ES03_73

value: ES03_73 human embryonic stem cell mRNA


title: NSCB_expression_380K_H1

value: H1 human embryonic stem cell mRNA


title: NSCB_expression_380K_HSF1_3


title: NSCB_expression_380K_HSF1_2


title: Stratagene Universal Human Reference vs. GM16243, 251209742752_A01

status: Public on Dec 08 2009

city: RTP

first: NIEHS

last: Microarray Core

state: NC

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

description: This study analyzes gene expression changes associated with a reduction of frataxin from peripheral blood of 28 children and 14 adults with Friedreich's ataxia compared to controls, as well as lymphoblastoid cell lines from patients.

hyb_protocol: 750ng each of Cy3 and Cy5 labeled cRNAs were fragmented and then hybridized for 17 hours in a rotating hybridization oven.

scan_protocol: Chips were washed and then scanned with an Agilent Scanner.

data_processing: The data were obtained with the Agilent Feature Extraction software (v9.1), using defaults for all parameters. The Feature Extraction Software performs error modeling before data are loaded into a database system. Images and GEML files, including error and P values, were exported from the Agilent Feature Extraction software and deposited into Rosetta Resolver (version 5.0, build 5.0.0.2.48) (Rosetta Biosoftware, Kirkland, WA).  The ratios (Cy5/Cy3) were generated.

source_name: Stratagene Universal Human Reference

label_protocol: Starting with 500ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer’s protocol.

extract_protocol: Purchased Reference RNA

tag: Universal Human Reference total RNA from 10 human cell lines

label_protocol: Starting with 500ng of total RNA, Cy5 labeled cRNA was produced according to manufacturer’s protocol.

extract_protocol: Total RNA was prepared from total blood using QIAGEN Rneasy midi kit

tag: Lymphoblastoid cell line derived from 23 yo patient with Friedreich's ataxia; GAA = 670-1170


title: DNR treated vs non-treated 1h (replicate 3)

city: Barcelona

first: Benjamin

last: Pina

address: Jordi Girona, 18

country: Spain

institute: IDAEA-CSIC

hyb_protocol: Microarray prehybridization was performed in 5x SSC (SSC: 150 mM NaCl, 15mM Na-citrate, pH 7.0), 0.1% SDS, 1%BSA at 42ºC for 45 min. (Fluka, Sigma-Aldrich, Buchs SG, Switzerland). Labeled cDNA was dried in a vacuum trap and used as probe after  resuspension in 110 μl of hybridization solution (50% Formamide, 5xSSC, 0.1% SDS, 100 μg/ml salmon sperm from Invitrogen, Carlsbad, CA, USA). Hybridization and washing were performed in a Lucidea Slide Pro System (GE Healthcare, Uppsala, Sweden).

scan_protocol: GenePix 4000B fluorescence scanner and analyzed by Genepix® Pro 6.0 software (Axon Instruments, MDS Analytical Technologies, Toronto, Canada)

data_processing: Data was filtered according to spot quality. Only those spots with  intensities at least twice the background signal and with at least 75% of pixels with intensities above background plus two standard deviations were selected for further calculations. Following these  criteria, over 70% of spots in each array were usually found suitable for further analysis.

organism: Saccharomyces cerevisiae

source_name: Saccharomyces cerevisiae BY4741 strain

label_protocol: 20 µg of total RNA were used for cDNA synthesis and labeling with Cy3-dUTP and Cy5-dUTP fluorescent nucleotides, following indirect labeling protocol (CyScribe postlabeling kit, GE-Healthcare). Labeling efficiency was evaluated by measuring Cy3 or Cy5 absorbance in Nanodrop Spectrophotometer.

growth_protocol: S. cerevisiae (BY4741 erg6Δ (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YML008c::KanMX4,  from EUROSCARF, Frankfurt, Germany) was grown in 500 ml of YPD medium at 30°C in an environmental shaker (250 rpm) until OD=0.4

extract_protocol: Total RNA extracted by RiboPure RNA extraction kit (Ambion) following manufacturers instructions

treatment_protocol: The culture was divided into three aliquots and diluted four times with fresh YPD medium. Daunorubicin was added to each culture at a final concentration of 12 mM and cultures were allowed to grow for 1 or 4 hours. 500 mL aliquots were taken at times 0 (upon addition of daunorubicin), 1h and 4h.Untraated cultures were grown and samples in parallel

tag: strain

value: BY4741 strain carrying null mutation delta erg6

tag: reference

value: Untreated, incubated for 1h at 30C

tag: time point

tag: temperature

tag: agent

value: 12 mM daunorubicin


title: CHOP1497

status: Public on Dec 03 2009

city: Philadelphia

first: Jayanti

last: Jagannathan

state: PA

address: 34th & Civic center blvd

department: Oncology

institute: Childrens Hospital of Philadelphia

hyb_protocol: Illumina Human 6 expression Bead chip standard protocol

label: biotin

source_name: tumor RNA

extract_protocol: Trizol RNA extraction reagent protocol

tag: tissue type

value: neuroblastoma

value: primary tumor

tag: mycn status

tag: inss

tag: risk

tag: ploidy

tag: age

tag: shimada


title: TEL-AML1-SNP-#30-50kxba

status: Public on Sep 02 2009

city: Memphis

first: Charles

middle: G

last: Mullighan

state: TN

address: 262 Danny Thomas Place

department: Pathology

institute: St Jude Children's Research Hospital

description: 242 pediatric acute lymphoblastic leukemia blast samples were analyzed.

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

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

data_processing: CEL files were generated using GeneChip Operating Software v 3.0 and SNP calls generated using GeneChip DNA Analysis Software (GDAS) v 3.0 or Affymetrix GeneChip Genotyping Analysis Software (GTYPE) v 4.0.  The summary files (containing both expression values and SNP calls) were generated by dChipSNP, and a modified array normalization algorithm that accounts for gross cytogenetic abnormalities in the blast samples (described in the paper).

label: Biotin

source_name: Pediatric acute lymphoblastic leukemia (ALL) blast samples or reference leukemic blast samples.

extract_protocol: Genomic DNA was extracted from mononuclear cell preparations of leukemic blasts obtained from peripheral blood or bone marrow at diagnosis using the Qiagen DNA Blood Mini Kit. DNA quality and quantity was assessed using a Nanodrop Spectrophotometer, PicoGreen and agarose gel electrophoresis.

tag: Leukemic blasts from 242 pediatric acute lymphoblastic leukemia (ALL) patients, or reference leukemic blast samples used for DNA copy number analysis.


title: T-ALL-SNP-#15-250ksty