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| Status |
Public on Feb 09, 2014 |
| Title |
RNA-seq pro-B (c-Kit high) 12072 |
| Sample type |
SRA |
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| Source name |
Pro-B cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Pro-B cell
strain: C57BL/6
genotype: Ikzf1(fl/-) Cd79a(Cre/+)
chip antibody: n.a.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Rneasy Plus Mini Kit, Dynabeads mRNA purification kit
2-5 ng of cDNA, ChIP-precipitated DNA were used as starting material for the generation of single-end sequencing libraries as described by Illumina’s ChIP Sequencing sample preparation protocol. DNA fragments of the following sizes were selected for the different experiments: 200–350 bp for ChIPseq and 150–700 bp for RNA-seq. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were modified with uracil-N-glycosylase (New England Biolabs) unable to generate second strand. The barcode containing adaptor was used for tagging and PCR amplification. Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS library quantification kit. library construction protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
SuperScript® VILO™ cDNA Synthesis, strand-specific
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| Data processing |
Peak calling of ChIP-seq data and target gene assignment: Peaks were called using the MACS program version 1.3.6.1 (ref. 19) with default parameters, a read length of 36 for ChIP-seq and 76 for DHS-seq, a genome size of 2,654,911,517 bp (mm9) and the appropriate input control sample. Peaks were filtered for p-values of < 10-10. This stringent cutoff efficiently removed false positive (unique) peaks of technical replicas. Ikaros peaks were assigned to target genes exactly as described15. Ikaros Bio-ChIP-sequencing resulted in ~180 million reads. To make these data comparable with other, less deeply sequenced ChIP-seq data, we implemented a subsampling procedure as follows: Both the input and Ikaros ChIP-seq data were down-sampled randomly 10 times to 27.3 and 22.5 million reads, respectively. These read numbers were selected to match those of the DHS-seq data and previously used input sequences. Peaks were then called for all Ikaros ChIPseq and input subsample combinations (10x10), as described above. The 9878 Ikaros peaks, which were called in 80% of all combinations, were used for multiple peak overlap analysis based on the Multovl program20.
Analysis of RNA-seq data: For analysis of differential gene expression, the RNA-seq samples were cut down to a read length of 30 or 44 bp (depending on the RNA-seq experiments to be compared) and aligned to the mouse transcriptome using the TopHat version 1.4.1 (ref. 22). The number of reads per gene was counted using the HTseq version 0.5.3 (http://www-huber.embl.de/users/anders/HTSeq) with the overlap resolution mode option set to "union". As two independent RNA-seq experiments were available for each cell type analyzed in Fig. 3f-h and Fig. 8b,e, the corresponding RNA-seq data were compared by using the R package DESeq version 1.8.3 (ref. 23) to calculate the significance level of differential expression. To this end, the samples were normalized, and the dispersions were estimated using the default DESeq settings. Genes with an adjusted p-value of < 0.1 were called as differentially expressed.
Genome_build: MM9
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| Submission date |
Dec 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Meinrad Busslinger |
| E-mail(s) |
meinrad.busslinger@imp.ac.at
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| Organization name |
IMP
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| Lab |
Busslinger
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| Street address |
Campus-Vienna-Biocenter 1
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| City |
Vienna |
| ZIP/Postal code |
A-1030 |
| Country |
Austria |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE53595 |
Stage-specific control of early B cell development by the transcription factor Ikaros |
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| Relations |
| Reanalyzed by |
GSE80797 |
| BioSample |
SAMN02485398 |
| SRA |
SRX398241 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1296545_12072.rpkm.txt.gz |
281.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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