GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM783257 Query DataSets for GSM783257
Status Public on Sep 01, 2014
Title Th17
Sample type SRA
Source name Mouse Th17 cells
Organism Mus musculus
Characteristics strain: C57BL/6
age: 7 weeks to 4 months
tissue: spleen
cell type: T helper cell type 17 (Th17)
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 1
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 SRX093325
BioSample SAMN00714245

Supplementary file Size Download File type/resource
GSM783257_th17.sam.gz 699.1 Mb (ftp)(http) SAM
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap