|
| Status |
Public on Apr 22, 2015 |
| Title |
Tet2-/-_AML1_ETO_p2_Rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Kit-enriched hematopoietic bone marrow cells retrovirally transduced with AML1-ETO
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Tet2-/-
passage number: p2
|
| Treatment protocol |
To induce the CreER-mediated excision of Tet2, the cells were treated with 200 nM 4-OHT (Experimental) or EtOH (Control) for 2 passages (4 days). The cells were subsequently washed to remove residual 4-OHT and passaged every 2 days for a total of 10 to 20 passages.
|
| Growth protocol |
Primary Kit-enriched bone marrow cells from Tet2fl/fl;CreER mice were freshly transduced with MigR1-AE-IRES-GFP, sorted for GFP and plated in methylcellulose medium (M3534, StemCell Technologies). After three rounds of replating, the cells were shifted to grow in suspension in non-tissue culture treated plasticware in media containing 1× StemPro®-34 SFM (Invitrogen), 1x L-Glutamine, with SCF (CHO producer cell line), 10ng/ml IL-3 (Peprotech), 10ng/ml IL-6 (Peprotech) and 0.1mM 2-mercapto-ethanol. Primary hematopoietic cell lines expressing MLL-AF9 in the Tet2fl/fl;CreER background was generated and maintained in suspension media as previously described (Somervaille, T.C.P., and Cleary, M.L. (2006), Cancer Cell 10, 257–268)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Immature myeloid cells (GFP+, CD11b-, Gr1-, CD16/32+) were sorted from AML1-ETO cultures. Total RNA was isolated using the AllPrep DNA/RNA micro kit (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
100ng of total RNA of each sample was labeled using Ambion® WT Expression Kit (Ambion) according to manufacturers instructions
|
| |
|
| Hybridization protocol |
Affymetrix EukGE-WS2v4_450 hybridization protocol according to manufacturers instructions
|
| Scan protocol |
Cell intensity files (CEL files) were generated in the GeneChip Command Console Software (AGCC) (Affymetrix, Santa Clara, CA, USA)
|
| Description |
Gene expression data from Tet2-/-;AML1_ETO cells grown for 2 passages following Tet2 disruption
|
| Data processing |
Raw vaules RMA-normalized and summarized using Affymetrix Power Tools (APT). Unix command: apt-probeset-summarize -a rma -a rma-bg,quant-norm.sketch=-1.bioc=true.lowprecision=false.usepm=true,pm-only,med-polish.expon=true -a pm-only,dabg.chisq=true -p ${affy_dir}/MoGene-2_0-st.pgf -c ${affy_dir}/MoGene-2_0-st.clf -b ${affy_dir}/MoGene-2_0-st.bgp --qc-probesets ${affy_dir}/MoGene-2_0-st.qcc -m ${affy_dir}/MoGene-2_0-st.mps ${cel_dir}/*.CEL -o ${out_dir}/gene_level
|
| |
|
| Submission date |
Jul 18, 2014 |
| Last update date |
Apr 22, 2015 |
| Contact name |
Kasper Dindler Rasmussen |
| E-mail(s) |
[email protected]
|
| Organization name |
School of Life Sciences, University of Dundee
|
| Department |
Division of Molecular, Cellular, and Developmental Biology (MCDB)
|
| Street address |
Dow Street
|
| City |
Dundee |
| ZIP/Postal code |
DD1 5EH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16570 |
| Series (2) |
| GSE59586 |
Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis (Affymetrix) |
| GSE59591 |
Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis |
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