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Sample GSM783253 Query DataSets for GSM783253
Status Public on Sep 01, 2014
Title Naive_rep1
Sample type SRA
Source name Mouse Naive CD4+ T cells
Organism Mus musculus
Characteristics strain: C57BL/6
age: 7 weeks to 4 months
tissue: spleen
cell type: Naive CD4+ T cell
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.
Extracted molecule polyA RNA
Extraction protocol Illumina ChIP-seq sample prep
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer IIx
Description Biological replicate 1 of 2
Data processing Data is mapped to mm9 genome assembly using Tophat with parameters: -butterfly-search-solexal1.3-quals
Submission date Aug 20, 2011
Last update date May 15, 2019
Contact name Daniel Hebenstreit
Organization name University of Warwick
Street address Gibbet Hill Rd
City Coventry
ZIP/Postal code CV47AL
Country United Kingdom
Platform ID GPL11002
Series (1)
GSE31555 Integrating Genomics, Transcriptomics, and T-Cell Biology: An RNA-seq Atlas of the Murine CD4+ Transcriptome
Reanalyzed by GSE80797
SRA SRX093321
BioSample SAMN00714241

Supplementary file Size Download File type/resource
GSM783253_naive_rep1.sam.gz 655.5 Mb (ftp)(http) SAM
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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