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| Status |
Public on Sep 01, 2014 |
| Title |
nTreg_rep2 |
| Sample type |
SRA |
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| Source name |
Mouse nTreg cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 7 weeks to 4 months
tissue: spleen
cell type: Natural T regulatory cell (nTreg)
average fragment length: 400bp (standard deviation: 20bp)
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| Growth protocol |
Spleens of C57BL/6 mice aged from 7 weeks to 4 months were removed and softly homogenized through a nylon mesh. The medium used throughout the cell cultures was IMDM supplemented with 10 % FCS, 2 mM L-glutamine, penicillin, streptomycin and 50 mM b-mercaptoethanol. Cells were washed twice and purified using Ficoll density gradient centrifugation. CD4+CD62L+ cells were isolated by a two-step MACS purification using the CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec). Cells were seeded into 24 well plates that had been coated with a mix of anti-CD3 (1 mg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 mg/ml, clone 37.51, eBioscience) antibodies overnight, at a density of 250,000 cells/ml and a total volume of 2 ml. The following cytokines and antibodies, respectively, were added to the cultures: Th1, recombinant murine IL-12 (5 ng/ml, R&D Systems), Th2, recombinant murine IL-4 (10 ng/ml, R&D Systems) and neutralizing IFN-g (5 mg/ml, Sigma), Th17, recombinant human IL-6 (30 ng/ml, Immunotools), recombinant human TGF-beta (5 ng/ml, Sigma) and recombinant murine mIL-1beta (Immunotools). Cells were cultured for 4 to 5 days at 37C in 5 % CO2. After this, cells were taken away from the activation stimulus, diluted 1:2 in fresh medium containing the same cytokine concentration as before. RNA was prepared after 3 days resting time. Cell preparation for Treg cells was the same as for Th cells, with the following differences. The medium used throughout was RPMI supplemented with 10 % FCS, gentamycin and 10 mM b-mercaptoethanol. CD4+CD25- cells were isolated by negative depletion of lymphocytes stained with FITC-conjugated anti-CD8, anti-CD11b, anti-CD11c, anti-CD19, anti-Ly6G and anti-CD25 by MACS. For the generation of iTregs, cells were seeded out in anti-CD3e (0.6 ug/ml) coated plates at 200.000 cells per well of a 96-well plate and cultured in medium containing 20 ng/mL recombinant IL-2, anti-CD28 (4ug/ml) and rhTGF-beta (50ng/mL) for four days after which the RNA was prepared (no resting phase). For nTregs, the depletion step for CD4+ enrichment was followed by positive selection for CD25.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Illumina ChIP-seq sample prep
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Biological replicate 2 of 2
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| Data processing |
Data is mapped to mm9 genome assembly using Tophat with parameters: -r 328 -butterfly-search-solexal1.3-quals
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| Submission date |
Aug 20, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Daniel Hebenstreit |
| E-mail(s) |
D.Hebenstreit@warwick.ac.uk
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| Organization name |
University of Warwick
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| Street address |
Gibbet Hill Rd
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| City |
Coventry |
| ZIP/Postal code |
CV47AL |
| Country |
United Kingdom |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE31555 |
Integrating Genomics, Transcriptomics, and T-Cell Biology: An RNA-seq Atlas of the Murine CD4+ Transcriptome |
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| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX093329 |
| BioSample |
SAMN00714249 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM783261_ntreg_rep2.sam.gz |
977.3 Mb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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