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| Status |
Public on May 01, 2015 |
| Title |
WT1_LSK |
| Sample type |
SRA |
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| Source name |
bone marrow, LSK, WT
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype/variation: WT
cell type: LSK
donor age (years): 0.35
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| Treatment protocol |
Freshly dissected femurs and tibias were isolated from mice. Bone marrow was flushed with a syringe into RPMI 10%FCS media. Cells were passed through a 70um strainer. Red blood cells were lysed in ammonium chloride-potassium bicarbonate lysis buffer for 10 minutes on ice. Lineage depletion was performed using the EasySep® Mouse Hematopoietic Progenitor Cell Enrichment Kit. Granulocyte-Macrophage Progenitors (GMPs, lineage- c-Kit+ Sca-1-, FcγR+ CD34+) and LSK (lineage- cKit+ Sca-1-) were sorted on the FACS Aria. Antibodies used for flow cytometry were as follows: anti-Gr1 (Ly6G), anti-B220 (RA3-682), anti-CD34 (RAM34), FCR (93) anti-Sca-1 (D7), phospho-Stat5 from eBioscience; anti-c-Kit (2B8), anti-Mac-1 (CD11b) (M1/70), anti-NK1.1 (PK136), anti-Ter119 (Ter119,553673), anti-CD3 (145-2C11), CD45.1 (A20), CD45.2 (104) all from BD Biosciences); and anti-CD150 (TC-15-12F2.2) and CD48 (HM48-1; both from Biolegend. Both the bone marrow and spleen 'lineage cocktail' included anti-CD3, anti-Gr-1, anti-Mac-1 (CD11b), anti-NK1.1, anti-B220, and anti-Terr-119.
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| Growth protocol |
Cells were harvested from sacrificed animals treated as described in the methods section of the associated manuscript.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed directly in Trizol (Life Technologies).
Follow Ion Torrent protocol, start with 100ng RNA using Ambion Low Input RiboMinus Eukaryote System v2 kit + LifeTech Ion Total RNA seq kit V2.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
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| Description |
YOD-20
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| Data processing |
Raw fastq files were trimmed using cutadapt cutadapt (v1.2rc2) -f fastq -g GGCCAAGGCA -m 35 --too-short-output
Reads were mapped using TopHat (ver 2) -p 20 --library-type fr-secondstrand \ -G Mus_musculus.BCNIM37.67 --transcriptome-index transMM9/Ensembl -T mm9
Remaining unmapped reads were then aligned to the same ref as in prev step using bwa (ver 0.5.9) using default paramaters.
TopHat and BWA outputs were merged using Picard MergeSamFiles using default parameters.
Read counts were generated from mapped reads using HT-Seq count.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: Counts.txt file contains total raw read counts per gene from HT-Seq counts using mode = "union".
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| Submission date |
May 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeffrey Zhao |
| Organization name |
Memorial Sloan Kettering Cancer Center
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| Department |
Genomics Core
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| Street address |
1275 York Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL18635 |
| Series (1) |
| GSE57244 |
Tet2-/-Flt3ITD and WT stem and progenitor cells |
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| Relations |
| BioSample |
SAMN02742764 |
| SRA |
SRX531212 |
| 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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