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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 27, 2016 |
| Title |
8303 Flt3ITD/ITD,Dnmt3afl/- MxCre AML |
| Sample type |
SRA |
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| Source name |
primary AML
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| Organism |
Mus musculus |
| Characteristics |
tissue: cKit+ splenocytes
cell type: acute myeloid leukemia
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| Treatment protocol |
To isolated c-Kit+ leukemic stem/progenitor populations, freshly isolated single cells suspensions were incubated with CD117 MicroBeads and separated on an AutoMACS Pro separator (Miltenyi, San Diego, CA) according to manufacturer specifications. RNA was extracted from cKit+ purified splenocytes using TriZol (Invitrogen) according to manufacturers instructions.
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| Growth protocol |
Mice were euthanized with carbon dioxide and cervical dislocation. Spleens were harvested from moribund Flt3ITD/ITD;Dnmt3afl/- MxCre and Flt3ITD/ITD;Dnmt3afl/fl controls. Splenocytes were isolated from freshly sacrificed mice and crushed between two glass slides. Red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) buffer (Gibco), washed, and filtered to obtain single cells suspensions for downstream applications.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted according to Meyer et al., 2009. RNA quality and quantity were measured by Agilant Bioanalyzer. Then samples with a RIN>8.0 were processed by poly-A selection, stranded True-seq, paired-end 75 base pair (bp), 40 million reads.
RNAs were detected by RNAseq analysis using paired-end 75 nucleotide (nt) sequencing and 40 million reads for all samples. RNA Seq libraries were generated with Illumina TruSeq Stranded mRNA HT kits.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
AML experimental mouse #8303 ckit+ splenocytes
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| Data processing |
FASTQ sequence files were aligned to the mouse genome version 10 (mm10) using TopHat2 with de novo junction discovery and separately to mm9 using RSEM.
The fastq files were aligned to mouse genome mm10 using BowTie 2 and TopHat 2 (Kim et al., 2013; Trapnell et al., 2009). BAM files were further processed in AltAnalyze 2.0.9 (Emig et al., 2010; Salomonis et al., 2009; Salomonis et al., 2010). To identify differentially expressed genes using default parameters moderated t-test with Benjamini-Hochberg multiple testing correction, raw p-value cut-off of 0.05, and an expression cut-off of 2-fold. RSEM gene-expression TPM values were all subjected to the same defaults in AltAnlayze for statistical analysis.
For all RNAseq experiments pathways and ontology enrichment analyses were performed with ToppGene (Chen et al., 2009) and GO-Elite (Zambon et al., 2012). All heatmaps from AltAnalyze were created using the HOPACH package via a connection to R (Salomonis et al., 2005). All gene set enrichment analyses were performed using GSEA v.2.2.0 (Mootha et al., 2003; Subramanian et al., 2005).
Genome_build: mm9 and mm10
Supplementary_files_format_and_content: tab-delimited text files
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| Submission date |
Feb 11, 2016 |
| Last update date |
Mar 01, 2022 |
| Contact name |
Nathan Salomonis |
| E-mail(s) |
nathan.salomonis@cchmc.org
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| Organization name |
Cincinnati Children's Hospital
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| Department |
Biomedical Informatics
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| Lab |
Nathan Salomonis
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| Street address |
3333 Burnet Avenue
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| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE77846 |
Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia (Bulk RNA-Seq) |
| GSE77849 |
Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia |
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| Relations |
| Reanalyzed by |
GSE192639 |
| SRA |
SRX1571631 |
| BioSample |
SAMN04501495 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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