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Sample GSM1119587 Query DataSets for GSM1119587
Status Public on Dec 31, 2013
Title MBD-Treg
Sample type SRA
Source name regulatory T cells
Organism Mus musculus
Characteristics cell type: regulatory T cells
surface marker: CD3+CD4+CD25++
strain: Balb/c
Sex: female
Treatment protocol Ex vivo-isolated spleen and lymph node cells were magnetically enriched for CD4+ cells by using the Miltenyi Biotech autoMACS system. The cells were stimulated for 3 hours by PMA (10ng/ml)/Ionomycin (500ng/ml) and then labelled by using a cytokine secretion assay for IL-17A and Interferon g (Miltenyi Biotech). All procedures were performed according to the manufacturers protocols. For purification of regulatory T cells we first sorted CD3+CD4+CD25++ cells by FACS and stimulated them finally in the presence of PMA/Ionomycin for 3 hours.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA from FACS-sorted T cell subsets (see markers above) was isolated by using the NucleoSpin Tissue XS kit (Macherey-Nagel).
Fragmentation of genomic DNA was performed on a Covaris S2. The size of the fragments was controlled with an Agilent 2100 system (Agilent Technologies). Methylated DNA was enriched by using a MethylCap kit (Diagenode AF-100-0048, Belgium). The procedure was done according to the manufacturer's protocol. We used a modified multiplexed paired end ChIP protocol (Illumina) to generate a library for next generation sequencing from 250ng precipitated DNA. Briefly, we used the DNA Sample Prep Master Mix Set 1 (New England Biolabs, E6040) in combination with the Multiplexing Sample Preparation Oligo Kit (Illumina, PE-400-1001). The preparation was performed on an Apollo 324 (IntegenX) with PrepX DNA or PrepX-32-DNA Library Kit (400007; 400021) according to the manufacturer's protocol. Small DNA fragments were removed from the samples by separation on a 2% agarose gel. Fragments between 250 and 350bp were excised and purified by using a Gel Extraction Kit (Qiagen, No. 28704).
Library strategy MBD-Seq
Library source genomic
Library selection MBD2 protein methyl-CpG binding domain
Instrument model Illumina HiSeq 2000
Data processing Illumina Casava1.8.0 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, subjected to FASTQC quality control tool 0.10.0.
Sequenced reads were then mapped to NCBI37/mm9 whole genome using bowtie v0.12.7 with 0 mismatches in the seed.
Only unique paired reads were retained and both fragments must be located within 400bp of each other on the reference genome.
Fragments that were identical but mapped into the same region were translated into an activity value. The activity value was used to classify the grade of methylation.
Genome_build: mm9
Supplementary_files_format_and_content: File 'Methylation_T_cell_subsets.xls' contains region ID, gene ID, chromosomal location, CpG content, activity and classification into methylated and non-methylated in Tnaive, Th1 or Th17 cells.
Supplementary_files_format_and_content: File 'TH17_vs_T_cell_subsets.txt' contains region ID, gene ID, gene label, chromosomal location, loci classification and activity in Tnaive, Th1 cells, Th17 cells and Tregs. The list of 10000 regions was filtered for no activity in Th17 cells but in all other T cell subsets. Integration of Treg MBD-seq to effector T cells required a normalization, so that every value is normalized on the total amount of mapped reads.
Submission date Apr 09, 2013
Last update date May 15, 2019
Contact name Stefan Floess
Organization name Helmholtz-Centre for Infection Research
Department Experimental Immunology
Street address Inhoffenstr. 7
City Braunschweig
ZIP/Postal code 38124
Country Germany
Platform ID GPL13112
Series (1)
GSE45911 Methylome of CD4+ T cell subsets
SRA SRX263392
BioSample SAMN02028519

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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