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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 27, 2016 |
| Title |
wtGMP_3 |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue type: bone marrow
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| Treatment protocol |
Isolated bone marrow cells were first stained with lineage cocktail antibodies against Cd5, Cd11b, Cd45R/B220, Ly-6G (Gr-1) and Ter119 (R&D Systems) and counter selected using magnetic beads. Cells were then stained with streptavidin Pacific Blue conjugated, biotinconjugated FcγRII/III, Alexa Fluor 647-conjugated Cd34, allophycocyanin-conjugated c-Kit and phycoerythrin-Cy7-conjugated Sca-1 monoclonal antibodies. The following sorting scheme were applied for FACS experiments: Granuolocyte macrophage progenitor cells (GMPs): IL-7Rα-, Lin- ,Sca-1-, c-Kit+, Cd34+, FcγRII/III-high; Long-term hematopoietic stem cells (LT-HSCs) Lin-, Sca-1+, c-Kit+, Cd34-, Cd48-, Cd150+
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| Growth protocol |
The cDNA of human MLL-AF9 fusion gene (a kind gift from J. Hess, University of Michigan, USA) was subcloned into the pLox-Cre vector backbone. 20 μg of p2Low-MLL-AF9 vector DNA were electroporated into the A2Lox-cre targeting cells pretreated with doxycycline in order to active Cre recombinase and then selected using 300 μg/ml G418. Selected clones were used to generate chimeric transgenic lines using the aggregation method and standard protocols. Chimeric mice were backcrossed with C57BL/6 for up to 10 generations. Double heterozygous female mice were identified using genotyping PCR and then used for subsequent animal experiments.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Arcuturus PicoPure RNA Isolation kit (and DNAse treated according to the kits manual.
RNA-seq libraries were prepared with the ScriptSeq Complete Kit (Human/Mouse/Rat) - Low Input (EpiCentre) starting from 150ng total RNA.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
BSSE_QGF_14038
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| Data processing |
HiSeq 2000 was run with HCS 2.0.12 and base calling was performed with RTA 1.17.21.3
The 3′ adaptor was removed by aligning it to the read allowing one or two mismatches in prefix alignments of at least seven or ten bases, respectively. Low-complexity reads were filtered out based on their dinucleotide entropy. All the reads that were shorter than 14 nucleotides were removed. Alignments to the Mus musculus genome (May 8, 2009, http://www.sanger.ac.uk/Projects/S_pombe/) were performed by the software bowtie (version 0.9.9.1) ( Langmead et al., 2009) with parameters -v 2 -a -m 100, tracking up to 100 best alignment positions per query and allowing at most two mismatches. To track genomically untemplated hits (e.g., exon-exon junctions or missing parts in the current assembly), the reads were also mapped to an annotation database containing known sequences (http://www.sanger.ac.uk/Projects/S_pombe/). In that case, all best hits with at most two mismatches were tracked. Each alignment was weighted by the inverse of the number of hits. In the cases where a read had more hits to an individual sequence from the annotation database than to the whole genome, the former number of hits was selected to ensure that the total weight of a read does not exceed one. Genomic read coverage plots were based on weighted alignments.
2007 M. musculus genome assembly (mm9) was used as a basis for all analyses. Low-complexity reads were filtered out based on their dinucleotide entropy (removing <1% of the reads). Alignments to the M. musculus genome were performed by the software bowtie (Langmead et al. 2009) with parameters -v 2 -a -m 100, tracking up to 100 best alignment positions per query and allowing at most two mismatches.
Reads mapping to mouse RefSeq mRNAs (fasta sequences downloaded from http://www.ncbi.nlm.nih.gov/nuccore on 2012-08-13) were counted per gene and per sample
Genome_build: mm9
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| Submission date |
Jan 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Antoine Peters |
| E-mail(s) |
[email protected]
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| Organization name |
Friedrich Miescher Institute for Biomedical Research (FMI)
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE65383 |
MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome [RNA-Seq] |
| GSE65384 |
MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome |
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| Relations |
| BioSample |
SAMN03299274 |
| SRA |
SRX857078 |
| 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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