MAGE-TAB Version	1.1							
Investigation Title	TARGET ALL SNP Array Profiling of DNA Copy Number Alterations in the Children’s Oncology Group AALL0232 and AALL0331 Cohorts							
Experimental Design	genotyping design	disease state design						
Experimental Design Term Source REF	EFO	EFO						
Experimental Factor Name								
Experimental Factor Type								
Experimental Factor Term Source REF								
Person Last Name	NCI Office of Cancer Genomics (OCG)	NCI Center for Biomedical Informatics and Information Technology (CBIIT)	Song	Mullighan	Hunger			
Person First Name			Guangchun	Charles	Stephen			
Person Mid Initials					P			
Person Email	ocg@mail.nih.gov	ncicbiit@mail.nih.gov	guangchun.song@stjude.org	charles.mullighan@stjude.org	hungers@chop.edu			
Person Phone	+1 301 451 8027	+1 888 478 4423		901-595-3387				
Person Fax	+1 301 480 4368			901-595-5947				
Person Address	31 Center Dr, Rm 10A07, Bethesda, MD 20892	9609 Medical Center Dr, Rockville, MD 20850		262 Danny Thomas Place, Mail Stop 342, Memphis TN 38105	3401 Civic Center Blvd Philadelphia, PA 19104			
Person Affiliation	National Cancer Institute	National Cancer Institute	St Jude Children’s Research Hospital	St Jude Children’s Research Hospital	Children's Hospital of Philadelphia			
Person Roles	funder	data coder;curator	data analyst;submitter	investigator	investigator			
Person Roles Term Source REF	EFO	EFO;EFO	EFO	EFO	EFO			
Quality Control Type								
Quality Control Term Source REF								
Replicate Type								
Replicate Term Source REF								
Normalization Type								
Normalization Term Source REF								
Date of Experiment								
Public Release Date								
PubMed ID								
Publication DOI								
Publication Author List								
Publication Title								
Publication Status								
Publication Status Term Source REF								
Experiment Description								
Protocol Name	stjude.org:Protocol:DNA-Extraction-Qiagen-DNAeasy:01	stjude.org:Protocol:DNA-Extraction-Phenol:01	stjude.org:Protocol:CopyNumberArray-Labeling-Affymetrix-SNP6:01	stjude.org:Protocol:CopyNumberArray-Hybridization-Affymetrix-SNP6:01	stjude.org:Protocol:CopyNumberArray-Scanning-Affymetrix-SNP6:01	stjude.org:Protocol:CopyNumberArray-DataNormalization-Birdseed:01	stjude.org:Protocol:CopyNumberArray-DataNormalization-dChipPounds:01	stjude.org:Protocol:CopyNumberArray-CnvSegment:01
Protocol Type	nucleic acid extraction protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol 	array scanning protocol	normalization data transformation protocol	normalization data transformation protocol	data transformation protocol
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Protocol Description	"Qiagen columns DNAeasy or DNA blood mini using the manufacturers protocol"	"Prelabel tubes with a membrane marking pen (ink is not washed off by alcohol): Lawson #133471.
"
Thaw or harvest cells
Pellet cells in 5x10^6 aliquots (e.g. 10,000g for 1 min in a microfuge)
Wash once in PBS (Any 1x PBS is OK, e.g Lonza 17-516F), pellet

Resuspend in 480ul DNA lysis buffer
 10mM Tris       (5mL of 1M Tris)
 10 mM NaCl    (1mL of 5M NaCl)
 10 mM EDTA (50mL of 100mM EDTA)  qs to 500mL volume, pH 8.0

Add 12.5 ul 20% SDS (Fisher BP1311-200) 
Add 10 ul proteinase K (Roche 03115887001)
Invert 20-30 times
Incubate at 37 deg o/night in a waterbath, or for 2 hrs at 55 deg

Place a 15ml Falcon filled with 100% Ethanol [Pharmacy] in a -80, or on dry ice.
Add 5ul RNAseA (Qiagen #19101, Lawson 111235). Invert 50x.
Incubate at 37deg for 10min
Add 10ul 5M NaCl 
Add 1ul Glycogen carrier [ Lawson 110901; Roche/Boeringer #901393, $81.18]

Go to fume hood
Swirl to mix Buffer Saturated Phenol [Invitrogen 15513-039 100ml; 15513-047 400ml]
Add 500ul phenol per sample. Mix by vigorous inversion 30-50x to achieve a homogeneous milky solution
Spin 16000g 5min

With a 1000ul pipette, carefully aspirate the top aqueous phase trying not to disturb the interphase and dispense into a new prelabelled 1.5 ml tube. Note: at this stage, if it was a generous cell pellet, the aqueous phase may be very viscous and you may aspirate some of the interphase – don’t worry.

Add 500ul PCI (Phenol:Chloroform:Isoamyl alcohol, Invitrogen 15593-031, 100ml; 15593-049 400ml) and mix/shake as above.
Centrifuge 16000g for 3 min.
Aspirate aqueous phase into a new tube. If still very viscous/goopy, repeat this PCI extraction one more time.

Add 1 ml cold ethanol (ie to fill the tube leaving just enough air to enable mixing).
Invert 50x.  Vortex for 3-5 seconds.  You should see a nice aggregate of DNA.

Spin at 4 degrees for 10 min at 20,000g in a centrifuge. Remove ethanol by aspiration.
Wash once with (~600-800uL) 70% ethanol. Scrape tube on rack to dislodge the pellet. Invert several times to wash the pellet.
Spin (4 deg) at 20,000g for 5 min to pellet.
Remove ethanol in two stages: aspirate most of the ethanol with a 1000ul pipette. Spin briefly to 16000g, then remove residual ethanol with a 100-200ul pipette.
Air dry for 10min. DO NOT OVERDRY – MAKES RESUSPENSION DIFFICULT.

Resuspend in 200ul DNA hydration solution (100ml Qiagen Cat #158914, 500ml Cat #158916)
Add the solution, then incubate at 55 degrees for 2 hours. Gently pipette to loosen the pellet.
Then incubate overnight at 37.
Note: the DNA is typically quite viscous and it is best to wait at least overnight, or longer at 4 degrees to fully resuspend before ODing.
QC by spectrophotometry (e.g. Nanodrop) and by electrophoresing 50-100ng in a 0.8% agarose gel.
Then store at 4 degrees.

Stock Solutions for DNA Lysis Buffer:
For 500mL of 1M Tris:  Add 60.57g of Tris bring to 500mL volume with distilled H20.  (Sigma #T6066-1KG; Lawson #107591)
For 200mL of 5M NaCl:  Add 58.44g of NaCl bring to 200mL volume with distilled H20.
For 500mL of 100mM EDTA:  Add 18.61g of EDTA bring to 500mL volume with distilled H20. (Sigma #E5134-500G; Lawson #112812)
	Affymetrix manufacturer's standard protocol	Affymetrix manufacturer's standard protocol	Affymetrix manufacturer's standard protocol	Affymetrix SNP6 genotypes were generated using the birdseed v2 algorithm in Genotyping Console (Affymetrix). Samples that failed standard QC metrics (contrast QC) were excluded.
	To generate copy number data, data were analyzed using a extensively used and validated algorithm developed at St Jude Children’s Research Hospital.  Affymetrix SNP array CEL files and SNP call files (either .CHP or .TXT files) were imported into dChip (http://www.hsph.harvard.edu/cli/complab/dchip/; Lin M, Wei LJ, Sellers WR, Lieberfarb M, Wong WH, Li C. dChipSNP: significance curve and clustering of SNP-array-based loss-of-heterozygosity data. Bioinformatics 2004;20:1233-40) and probe level values summarized. Data were exported and normalized using the reference normalization algorithm (Pounds S, Cheng C, Mullighan C, Raimondi SC, Shurtleff S, Downing JR. Reference alignment of SNP microarray signals for copy number analysis of tumors. Bioinformatics 2009;25:315-21). This algorithm uses user supplied or computationally detected diploid chromosomes to guide normalization of the entire array on a sample-by-sample basis, and optimizes normalization of complex cancer samples while eliminating batch effects. This procedure generates the .cnmz file that includes both the summarize, normalized prove intensities and the genotype data.	Optimally normalized data were subjected to paired circular binary segmentation (Venkatraman ES, Olshen AB. A faster circular binary segmentation algorithm for the analysis of array CGH data. Bioinformatics 2007;23:657-63) with thresholds set to detect copy number segments of &gt;2.3 or &lt;.7 copies and at least 5 markers (250K data) or 8 markers (SNP6 data). Raw copy number segmentation results inspected and curated in dChip.
Protocol Parameters								
Protocol Hardware								
Protocol Software								
Protocol Contact								
SDRF File	TARGET_ALL_CopyNumberArray_Phase2_20160812.sdrf.txt							
Term Source Name	NCBITaxon	NCIt	MO	EFO	OBI			
Term Source File	http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://ncit.nci.nih.gov/	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo	http://purl.obolibrary.org/obo/obi			
Term Source Version								