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| Status |
Public on Nov 24, 2019 |
| Title |
RNA-seq_P3_WT1 |
| Sample type |
SRA |
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| Source name |
RNA-seq_P3_WT
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
genotype/variation: Erg fl/fl
tissue: Bone marrow
cell type: Progenitor B-cells
progenitor cell type: P2 (B220+, CD19+, CD93+, CD43-, CD25+)
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| Growth protocol |
Cells FACS-sorted from freshly harvested murine bone marrow
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| Extracted molecule |
total RNA |
| Extraction protocol |
Murine progenitor B-cells were FACS-sorted into RLT plus buffer (Qiagen) with 2-mercaptoethanol (Sigma-Aldrich, 10µl/mL RLT buffer). The RNA was extracted with Qiagen’s AllPrep micro kit.
cDNA was obtained using the NuGEN’s Ovation RNA-seq System V2, and libraries were prepared with NuGEN’s Ovation Ultralow System V2. All according to manufacturer’s instructions. The samples were subjected to 75-cycles sequencing on an Illumina NextSeq 500 instrument.
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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 |
The RNA-seq data was analyzed with bcbio-nextgen (https://github.com/chapmanb/bcbio-nextgen). Fastq files were aligned to the mm10 genome using STAR (Dobin et al., 2013, DOI:10.1093/bioinformatics/bts635). Transcript expression levels were estimated with Salmon (Patro et al., 2017, DOI: 10.1038/nmeth.4197). FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for QC metrics, and multiqc (Ewels et al., 2016, DOI: 10.1093/bioinformatics/btw354) for reporting. Data analysis was then performed with R/Bioconductor (https://www.R-project.org/, Gentleman et al. (2004, DOI: 10.1186/gb-2004-5-10-r80)). For the differential gene expression, we followed the recommendations from Soneson et al. (2015, DOI: 10.12688/f1000research.7563.2). Gene expression levels were assessed by adding all the transcript levels for a given gene and normalization of the counts was done with the lengthScaledTPM function of the tximport package followed by a limma-voom procedure: for each population (P1-3), genes with CPM>1 in at least 2 samples were selected, the TMM normalization was run using the calcNormFactors function of the limma package (Ritchie et al., 2015, DOI: 10.1093/nar/gkv007), and a linear model with Erg∆/∆ vs. Ergfl/fl was fitted (voom / lmFit / eBayes). P-values were adjusted using the Benjamini-Hochberg procedure.
Genome_build: mm10
Supplementary_files_format_and_content: Excel sheet containing differential expression analysis, Erg fl/fl;CD2iCre (KO) vs. Erg fl/fl (WT). A separate tab is provided for the analysis of each of the three progenitor populations (P1, P2, P3) and an additional tab explains the content of each column.
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| Submission date |
Mar 28, 2019 |
| Last update date |
Nov 24, 2019 |
| Contact name |
Bo Porse |
| Organization name |
University of Copenhagen
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| Department |
Finsen Laboratory and BRIC
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| Lab |
Porse lab
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| Street address |
Ole Maaløes Vej 5, 4
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| City |
Copenhagen |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE128977 |
ERG controls B-cell development by promoting Igh V-to-DJ recombination [RNA-seq] |
| GSE128978 |
ERG controls B-cell development by promoting Igh V-to-DJ recombination |
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| Relations |
| BioSample |
SAMN11279406 |
| SRA |
SRX5587697 |
| 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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