Illumina HiSeq 2500 paired end sequencing; GSM2060320: Cell1-A1 cKit+ Flt3ITD/ITD,Dnmt3afl/- MxCre  AML-1; Mus musculus; RNA-Seq

Cell1-A1 cKit+ Flt3ITD/ITD,Dnmt3afl/- MxCre AML-1

Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human acute myeloid leukemia (AML) cases with a 5-year overall survival of approximately 30%. In CN-AML with poorer prognosis, mutations in the de novo DNA methyltransferase (DNMT3A) and the FMS-like tyrosine kinase 3 (Flt3) commonly co-occur (1-3). We demonstrate that mice with Flt3-internal-tandem duplication (Flt3ITD) and inducible deletion of Dnmt3a spontaneously develop a rapidly-lethal, completely-penetrant, and transplantable AML of normal karyotype. These murine AML retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3-ITD/DNMT3A-mutant primary human and murine AML demonstrate a similar pattern of global DNA methylation. In the murine model, rescuing DNMT3A expression was accompanied by DNA re-methylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible.  Differentially methylated genomic regions were associated with changes in the expression of nearby genes. Moreover, dissection of the cellular architecture of the AML model using single-cell RNA-Seq, flow cytometry and colony assays identified clonogenic subpopulations that differentially express genes that are sensitive to the methylation of nearby genomic loci and varied in response to Dnmt3a levels. Thus, Dnmt3a haploinsufficiency transforms Flt3ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation. Overall design: To identify the gene expression changes associated with Dnmt3a loss of function in human and murine Flt3-ITD and Dnmt3a-mutant AML (Single Cell RNA-Seq).

Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia (Single Cell RNA-Seq)

Submitted by Gene Expression Omnibus on 29-MAR-2016

Single CD117+ (c-Kit+) cells were prepared using the C1™ Single-Cell Auto Prep System (Fluidigm, San Fransisco, CA), according to the manufacturer’s instructions.  In short, sorted cells were counted and resuspended at a concentration of 333,333 cells per 1 mL DPBS then loaded onto a primed C1 Single-Cell Auto Prep Integrated Fluidic Chip for mRNA-Seq (5-10 µm). After the fluidic step, cell separation was visually scored to identify wells containing single live cells. In two independent rounds, 52 and 56 cells were captured. Cells were lysed on the chip and reverse transcription was performed using the Clontech SMARTer® Kit with the mRNA Seq: RT + Amp (1771x) according to the manual. After the reverse transcriptase step, cDNAs were transferred to a 96 well plate and diluted with 6 µl C1™ DNA Dilution Reagent. cDNAs were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island, NY) and Agilent High Sensitivity DNA Kit (Agilent Technologies (Santa Clara, CA). Libraries for the first 48 visually validated cells from each of the two independent captures were prepared using the Nextera XT DNA Library Preparation Kit (Illumina Inc, Santa Clara, CA).  In each single-cell library preparation, a total of 125pg cDNA was tagmented at 55 °C for 20 minutes. Next, libraries were pooled and purified on the AMPure® bead-based magnetic separation Kit prior to a final quality control check using both the Qubit® dsDNA HS Assay Kit (Life Tecnologies, Grand Island, NY) and Agilent High Sensitivity DNA Kit. The majority of cDNA fragments were confirmed to be between 200-602 bp, qualifying the libraries for sequencing. All 96 single-cell RNA-Seq libraries were sequenced on one HiSeq 2500 gel.

Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia (Single Cell RNA-Seq)

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