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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 21, 2014 |
| Title |
Egr2CKO CD4+ T 4h RNASeq |
| Sample type |
SRA |
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| Source name |
T cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Egr2-/-
cell type: T cells
tissue: Spleen
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| Treatment protocol |
For RNA-Seq,naïve CD4+ T cells, not stimulated or stimulated with anti-TCR for 1, 4, and 16 h
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| Growth protocol |
Naïve CD4+ T cells from spleen and lymph nodes were purified from 6 week old mice using a CD4+CD62+ T cell Isolation Kit II (Miltenyi). Cells were activated with 2 μg/ml plate-bound anti-CD3 + 1 μg/ml soluble anti-CD28 (PharMingen)
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-Seq, total RNA was extracted from 5x10^6 cells per sample using Rneasy Mini kit (QIAGEN) and the quality and concentration were determined by Nanodrop ND-1000 spectrophotometer. Double-stranded cDNA was synthesized using random hexamer primers, SuperScript II, DNA polymerase I, and T4 DNA polymerase (all from Invitrogen) and fragmented using Bioraptor. After repairing ends, adaptor (Illumina) was added using T4 DNA ligase (New England Biolabs), and 250-450 bp fragments were isolated using 2% E-Gel (Invitrogen) and amplified for 17 cycles using PE 1.0 and PE 2.0 primers (Illumina) and Phusion High Fidelity PCR Master Mix (New England Biolabs).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
TCR stimulated
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| Data processing |
For ChIP-Seq and RNA-Seq, Basecalls performed using CASAVA 1.8 (HiSeq2000)
Raw reads were aligned to the mm9 mouse genome assembly using Bowtie 0.12.7
Aligned reads are converted to BED format and removed redundancies using unpublished python and perl scripts
For RNA-Seq, Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using mm9 refseq database
Genome_build: mm9
Supplementary_files_format_and_content: BED files are generated using BEDTOOLS and unpublished python scripts, rpkm (abundance measurement) files are generated using definition in [Mortazavi et al., 2008] and unpublished python scripts
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| Submission date |
Jul 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peng Li |
| E-mail(s) |
[email protected]
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| Organization name |
NIH
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| Department |
NHLBI
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| Lab |
LMI
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE49366 |
EGR2 is Critical for Peripheral Naïve T Cell Differentiation and the T-cell Response to Influenza |
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| Relations |
| BioSample |
SAMN02297747 |
| SRA |
SRX329264 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1198174_WJL2012-302-mm9-Egr2-CD4-4h.txt.gz |
550.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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