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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

E-mail(s)

stefan.floess@helmholtz-hzi.de

Organization name

Helmholtz-Centre for Infection Research

Department

Experimental Immunology