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| Status |
Public on Jan 22, 2019 |
| Title |
MPP7_Tet2KO_MO182 [RNA-Seq] |
| Sample type |
SRA |
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| Source name |
Hematopoietic cells sorted from bone marrow
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| Organism |
Mus musculus |
| Characteristics |
cell type: Multipotent Progenitors (MPPs), Lin-cKit+Sca1+CD48+CD150-
genotype: Tet2-/-
age at isolation: 40 weeks (10mo)
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| Treatment protocol |
Single-cell suspensions of mouse bone marrow were erythrolysed, enriched for Kit expression (CD117 microbeads, Miltenyi Biotech) and stained with antibodies against surface markers: Lineage (B220-PECy5 (RA3-6B2, eBioscience), CD11b-PECy5 (M1/70, eBioscience), Ter119, PECy5 (TER-119, eBioscience), CD3e-PECy5 (145-2C11, eBioscience), Gr1-PECy5 (RB6-8C5, eBioscience)), Sca1-BV421 (D7, BD biosciences), cKit-AlexaFlour 780 (2B8, eBioscience), CD150-APC (TC15-12F12.2, Biolegend), CD48-PE (HM48-1, eBiocience), CD16/32-PECy7 (93, eBioscience). The following combination of surface markers was used to define hematopoietic progenitor populations: Multipotent Progenitors (MPPs), Lin-cKit+Sca1+CD48+CD150-; Granulocyte-Monocyte Progenitors (GMPs), Lin-cKit+Sca1-CD150-CD16/32+.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from FACS purified hematopoietic cell populations using RNeasy Micro Kit (Qiagen). Quality assessment of total RNA was performed using an RNA 6000 Pico Kit (Agilent) and only samples with a RNA integrity number above 8 were used for further analysis. RNA concentrations were determined using a Qubit RNA HS assay kit (ThermoFisher Scientific) and a Qubit 2.0 fluorometer.
A proportion of total RNA (2 ng) isolated from four biological replicates (individual mice with wildtype or Tet2-deficient hematopoiesis) was amplified and size-selected with the Ovation RNA amplification system v2 (NuGen, Cat: 7102) and sequencing adaptors were added to the resulting cDNA using the Ovation Ultra Low v2 system (NuGen) according to manufacturer´s instructions. RNA-seq libraries were quality checked using a DNA 1000 Kit (Agilent) and sequenced using an Illumina NextSeq 550 instrument (75bp single-end).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
Differential gene expression analysis was performed with the RNA Express 1.0 workflow application (Illumina). Briefly, raw sequencing reads were adaptor trimmed and aligned to the mouse genome (mm10) with STAR aligner. Mapped reads were assigned to genes and differential gene expression was analysed using DESeq2. Four biological replicates were used for the DESeq2 analysis. Lowly expressed genes (mean count lower than 10) as well as outlier samples (based on DESeq2 variance model) were excluded and marked in the "status" column. Supplementary files: TAB-delimited files containing the final output tables of differentially expressed genes in MPP and GMP cells, respectively. The data is organised in columns with the titles: Gene, Status, baseMean, Log2FoldChange, lfcSE (log2 fold change standard error), p-value, p-adj (multiple correction adjusted p-value).
Genome_build: mm10
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| Submission date |
Jun 18, 2018 |
| Last update date |
Jan 22, 2019 |
| Contact name |
Kasper Dindler Rasmussen |
| E-mail(s) |
[email protected]
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| Organization name |
School of Life Sciences, University of Dundee
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| Department |
Division of Molecular, Cellular, and Developmental Biology (MCDB)
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| Street address |
Dow Street
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| City |
Dundee |
| ZIP/Postal code |
DD1 5EH |
| Country |
United Kingdom |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE115967 |
TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells [RNA-Seq] |
| GSE115972 |
TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells |
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| Relations |
| BioSample |
SAMN09444890 |
| SRA |
SRX4232583 |
| 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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