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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 27, 2015 |
| Title |
RNA-seq TH-ProB cultured 1 day on OP9, rep3 |
| Sample type |
SRA |
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| Source name |
Ebf1+/- Pax5+/- ProB, 1 day culture on OP9 stromal cells
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6J
genotype/variation: Ebf1+/- Pax5+/-
cell type: ProB
treatment: 1 day culture on OP9 stromal cells
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| Treatment protocol |
See above.
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| Growth protocol |
ProB-cell isolation / culture protocol: For sorting of Pro B cells, CD16/CD32 (FC) -blocked (93, eBioscience) cells were stained with antibodies against lineage markers CD11b/Mac1 (M1/70), Gr1 (RB6-8C5), TER119 (Ter119), CD3 (17A2, BD Pharmingen), CD11c (N418) and NK1.1 (PK136) and further stained with CD19 (ID3), CD45R/B220 (RA3-6B2), CD43 (S7), IgM (RMM-1) and IgD (11-26. eBioscience, San Diego, CA). Cell sorting was performed on a BD FACSAriaTM (BD Biosciences, San Jose, California) using propidium iodide (PI, Invitrogen, Paisly, UK) as viability marker. For expansion, Pro-B cells were cultured on OP9 stromal cells supplemented with 10ng/mL KIT ligand, 10ng/mL Fms-like tyrosine kinase 3 ligand (FLT3L), and 10ng/mL Interleukin-7 in Optimem supplemented with 10% FCS, Beta-mercaptoethanol, HEPES and Penstrep.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA-Seq: RNA was extracted with the Qiagen RNeasy Micro Kit.
RNA-seq 1 day on OP9DL1 and OP9: Libraries were constructed using Nugen's Ovation Ultralow Library systems. Detailed protocol is available on their website. In short, input DNA were fragmented using sonication, end repaired, followed by ligation of adapters, amplification and finally bead purification. The samples were run on NextSeq 500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
For RNA-Seq samples, data analysis was performed with Arraystar (DNA STAR). The reads were aligned to mouse reference genome (mm9, NCBI 37) and RPKM normalized. Statistical analysis in Arraystar was performed with Student´s t-test with correction for multiple testing (Benjamini Hochberg). A p-value > 0.05 was considered as statistically significant.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: Processed data files include BED files. All genomic coordinates are relative to the mm9 mouse assembly.
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| Submission date |
May 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Rajesh Somasundaram |
| E-mail(s) |
[email protected]
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| Phone |
0046708890787
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| Organization name |
Linkoping University
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| Department |
Microbiology and Molecular Medicine
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| Lab |
Lab 1, Floor- 13 Dept of Clinical and Experimental Medicine (IKE)
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| Street address |
Dept of Clinical and Experimental Medicine (IKE)
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| City |
Linkoping |
| ZIP/Postal code |
SE-58185 |
| Country |
Sweden |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE69227 |
Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B-cell progenitors |
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| Relations |
| BioSample |
SAMN03734564 |
| SRA |
SRX1038497 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1695690_ProB_TH_OP9-3.bed.gz |
27.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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