Comment[ArrayExpressAccession]	E-MTAB-1764
MAGE-TAB Version	1.1
Investigation Title	IGR_U985_CLL_EGR2_GE_STUDY_OB
Comment[Submitted Name]	IGR_U985_CLL_EGR2_GE_STUDY_OB
Experiment Description	Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in western countries, is a clonal accumulation of mature B-lymphocytes and its natural history is yet unclear. By using sequencing and cellular biology approaches on a cohort of CLL patient samples, we show here that acquired CLL mutations are observed in hematopoietic multipotent progenitor fractions in the majority of patients. These early CLL mutations include recurrent inactivating mutations in NFKBIE (10.7%) and missense mutations in BRAF (3.6%) and EGR2 (8.3%). Functional analyses demonstrated that BRAF-G469R affects lymphoid differentiation and transforms the T-cell lineage in vivo. In addition, the EGR2 recurrent mutations were associated with transcriptional activation of EGR2 target genes in patients and cell cycle abnormality in cellular model. Our findings indicate that CLL may develop from an initial infra-clinic, pre-leukemic phase affecting immature hematopoietic cells. The present study concerns gene expression of mouse EML cell line transfected by EGR2 wt gene and 2 mutated forms  (E356K and H384N) and controls with the empty vector. Gene expression was performed in single color on Agilent 8x60K Mouse Genome array (design 028005).
Experimental Design	genetic modification design	replicate design	genotyping design
Comment[AEExperimentType]	transcription profiling by array
Comment[AEExperimentDisplayName]	Transcription profiling by array of mouse EML cell line transfected with EGR2 wt gene, 2 mutated forms (E356K and H384N) and controls with an empty vector
Comment[AdditionalFile:TXT]	028005_D_DNABack_BCLeft_20130207.txt
Experimental Factor Name	genotype
Experimental Factor Type	genotype
Quality Control Type	biological replicate
Quality Control Term Source REF	EFO
Public Release Date	2014-06-18
Person Last Name	DESSEN	BERNARD
Person First Name	Philippe	Olivier
Person Mid Initials
Person Email	dessen@gustaveroussy.fr	olivier.bernard@inserm.fr
Person Phone	33142114490
Person Address	INSERM U985, Institut Gustave Roussy, 114 rue E. Vaillant, 94805 Villejuif, Cedex, France	INSERM U985, Institut Gustave Roussy, 114 rue E. Vaillant, 94805 Villejuif, Cedex, France
Person Affiliation	CNRS	INSERM
Person Roles	submitter	investigator
PubMed ID	24920063
Publication Author List	Damm F, Mylonas E, Cosson A, Yoshida K, Della Valle V, Mouly E, Diop M, Scourzic L, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Kikushige Y, Davi F, Lambert J, Gautheret D, Merle-Beral H, Sutton L, Dessen P, Solary E, Akashi K, Vainchenker W, Mercher T, Droin N, Ogawa S, Nguyen-Khac F, Bernard OA.
Publication Title	Acquired initiating mutations in early hematopoietic cells of CLL patients.
Publication Status
Protocol Name	P-MTAB-33774	P-MTAB-33775	P-MTAB-33776	P-MTAB-33777	P-MTAB-33778	P-MTAB-33779
Protocol Type	growth protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning protocol	normalization data transformation protocol
Protocol Description	The growth factor dependant cell line EML 9 is a kind gift of Guy Mouchiroud (Lyon, France). Cells were grown in IMDM medium, 20% horse serum, 1% Penicilin/Streptomycine /Glutamine and supplemented with 10% of BHK cells supernatant. Twelve hours after transduction cells are washed, and seeded at 5X106 cells per well. Cells were counted and analysed by flow cytometry every 2 days. PE conjugated antibodies were Gr1 ( RB6-8C5), B220 (RA3-6B2)  from eBiosciences and cKit (2B8)  and CD117 from BD Pharmigen). Experiments were done at least twice in triplicate. Cell cycle was analysed 4 days after transduction using the BrdU Flow kit (BD Pharmigen, 552598). 7AAD staining was used for the determination of total DNA and APC conjugated anti BrdU for the determination of incorporated BrdU. Experiments were done at least twice in triplicate. Two days after transduction GFP-positive cells were flow- sorted and the RNA and protein were extracted using the RNA/DNA/Protein purification Plus Kit (47700, Norgen Biotek Corp). Protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. Anti-EGR2 (P100880, Aviva Systems Biology) and anti-Actine (A3853, Sigma) were used as primary antibodies. Secondary HRP conjugated antibodies (anti rabbit IgG (NA934V, GE), anti mouse IgG (NA931V, GE) and ECL Plus Kit (RPN2132, GE) were use for detection	RNA were extracted with Trizol protocol followed by Qiagen purification. The quantity and purity of the extracted RNA was evaluated using a NanoDrop spectrophotometer and its integrity measured using an Agilent Bioanalyzer	Agilent oligo Cy3 probes labelling protocol. Kit used for probe labelling: Agilent Low Input Quick Amp Labeling Kit, one-color (5190-2305) adapted for small amount of total RNA (100 ng total RNA per reaction). In 1.5 ml tubes add 100 ng total RNA from sample. Add 0.8 ul T7 promoter primer, 2ul Spike-in A for Cy3 labelling and add nuclease free water (Invitrogen ref:10977-015) to bring the volume to 5.3 ul. Denature by incubating at 65C for 10 minutes. Place the reactions on ice and incubate 5 min. Spin briefly. Prepare a Reverse Transcription master mix, adding for one reaction 2 ul First strand Buffer 5X, 1 ul DTT 0.1M, 0.5 ul dNTP 10 mM mix, 1.2 ul Affinity Script Block Mix. Master mix is prepared in batch for all the samples included in the study (vol per one reaction multiplied by number of samples. In each reaction tube containing denaturated RNA and T7 promoter primer in a volume of 5.3 ul, add 4.7 ul of Reverse Transcriptase master mix, and mix by gently pipetting. Incubate at 40C in a circulating water bath for 2 hours. Move samples to 65C for 15 minutes to inactivate enzymes, and incubate on ice for 5 minutes. Spin briefly. Prepare a in vitro transcription master mix, adding for one reaction : 0.75 ul Nuclease free water, 3.2 ul Transcription buffer 5X, 0.6 ul DTT 0.1 M, 1 ul NTP mix, 0.24 CTP-Cy3, 0.21 ul T7 RNA Polymerase Blend. Transcription master mix is prepared in batch for all the samples included in the study: (vol per one reaction multiplied by number of samples). To each RT reaction, add 6 ul Transcription master mix. Incubate at 40C for 2 hours. Add 84 ul nuclease free water and freeze at 20C. Labeled probes are purified using Qiagen Rneasy mini kit and protocol provided by Agilent. For each probe, add 350 ul RLT buffer, and 250 ul ethanol 100%. Mix by gently vortexing. Apply 700 ul on Rneasy columns and spin at 13,000 g for 30 s at 4C. Discard flow-through. Wash twice with RPE buffer. Dry the column and elute in 60 ul nuclease free water. Measure concentration and Cy3 incorporation using a Nanodrop spectrophotometer. Adjust concentration at 100 ng/ul. Freeze at 20C until hybridisation.	Agilent Hybridization Protocol (Chamber type: Agilent SureHyb Chamber; Quantity of labelled extract: 1650 ng per labelling; duration: 17 hours; volume: 100 ul per array; Temperature in C: 65). Add the following components in clean 1.5 ml tubes: 1650 ng linearly amplified cRNA labeled Cy3. Add 11 ul Bocking Agent 10X, 2.2 ul of Fragmentation buffer 25X and nuclease free water up to 55 ul. Mix by vortexing. Incubate at 60C for 30 minutes in dark. Spin briefly, add 55 ul of 2X hi-hybridization buffer. Mix gently. Assembly the Sure-Hyb hybridization chamber from Agilent. Place a backing slide with the plastic inner on upper side. Gently add 100 ul of 1x hybridisation solution and cover with the Agilent 8x60K (AMADID 28004) GE Human array properly oriented (active surface in contact with liquid). Finish to assembly the chamber and tight the screw. Hybridization was carried out for 17 hours at 65C in a rotating oven (Robbins Scientific, Mountain View, CA) at 10 rpm. The arrays were disassembled at room temperature in wash 1 buffer (Agilent), then washed 1 minute in a glass dish (Wheathon) at room temperature in wash 1 buffer, then 1 minute in wash 2 buffer at 37C and then 1 minute in acetonitril. Slides were dried using a nitrogen gun, and scanned by using an Agilent G2565 C DNA microarray scanner.	Scanning was performed with a Agilent G2565C DNA Microarray scanner using defaults parameters (100 PMT, 5 um resolution, at 20C in low ozone concentration environment. Microarray images were analysed by using Feature Extraction software version (10.7.3.1) from Agilent technologies and Agilent normalization protocol GE2_107_Sep09 with 028005_D_F_20130207 as design. Defaults settings were used.	A quantile array normalization was performed on raw gMedianSignal values with bioconductor LIMMA package. Controls and flags spots were filtered. Missing values were completed with KNN imputation method. Then the median of all probes for a given transcript was computed to summarize the data. Additional txt file provided see Comment[AdditionalFile:TXT] .
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO
Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MTAB-1764.sdrf.txt