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| Status |
Public on Apr 12, 2015 |
| Title |
5mC.Th17 |
| Sample type |
SRA |
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| Source name |
CD4+ T cells, 5mC.Th17
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell population: CD4+ T cells
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| Growth protocol |
Naive T cells were isolated by sorting CD4+CD25-CD62LhighCD44low cells from spleens and lymph nodes, differentiated under several Th cell conditions, and analyzed as described. The naive T cells (5 x 105 cells/well) were cultured at 37oC (5% CO2) in RPMI-1640 (Invitrogen Life Technologies). The medium was supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol. The cells were stimulated with the plate-bound a-CD3 (1 g/ml) and the soluble a-CD28 (1 g/ml). For Th0 differentiation, the cells were treated with 5 g/ml -IFN- (XMG1.2; BioXCell), 5 g/ml -IL-4 (11B11; BioXCell) and 30 U/ml IL-2. For Th1 differentiation, the cells were treated with 10 ng/ml mIL-12 (Peprotech), 5 g/ml -IL-4 and 30 U/ml IL-2. For Tr1 differentiation, the cells were treated with 50 ng/ml mIL-27 (R&D Systems), 2 ng/ml TGF- 1 (Peprotech) and 30 U/ml IL-2. When indicated, irradiated wild-type splenic antigen-presenting cells (APCs) were used for optimal Tr1 cell differentiation. For Th2 differentiation, the cells were treated with 10 ng/ml mIL-4 (Peprotech), 5 g/ml -IFN- . For iTreg differentiation, the cells were treated with 1 ng/ml TGF- 1 (Peprotech), 5 g/ml -IFN- , 5 g/ml -IL-4 and 30 U/ml IL-2. For Th17 differentiation, the cells were treated with 0.5 ng/ml TGF- 1, 10 ng/ml IL-6 (Peprotech), 5 g/ml -IFN- , and 5 g/ml -IL-4. When indicated, 10 ng/ml IL-23 and 10 ng/ml IL-1 (Peprotech) were used for optimal Th17 cell differentiation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
the genomic DNA was purified and sonicated. DNA fragments (4 ug) were denatured and incubated with antibodies against 5mC (Eurogentec) and 5hmC (Active Motif) at 4 degree overnight. Antibody-DNA complexes were captured by protein A agarose/Salmon sperm DNA. The immunoprecipitated DNA was purified. The results were normalized relative to the input control.
Libraries were prepared using Illumina ChIP-seq DNA preparation kit (IP-202-1012) according to the manufacture's instruction.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Reads obtained from two instruments: Illumina Genome Analyzer IIx and Illumina HiSeq 2500
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| Data processing |
Basecalls performed using CASAVA version 1.6 and 1.8.1
MeDIP-seq reads were aligned to the mm9 genome assembly using Bowtie 1.0.1 using -p 4 -v 2 -m 1
The unique reads for 5mC and 5hmC were mapped into non-overlapping 200 bp windows (400 bp gap size) of mouse genome (mm9). The peaks were called using SICER (Zang et al., 2009), with the Input DNA sample as a control and a FDR of 0.01 as the cutoff.
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using SICER.
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| Submission date |
Feb 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Raymond Doty |
| E-mail(s) |
rtdoty@u.washington.edu
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| Organization name |
University of Washington
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| Department |
Division of Hematology
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| Street address |
Box 357710
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE66268 |
Regulation of T cell cytokine expression by Tet2-mediated DNA demethylation [MeDIP-Seq] |
| GSE66945 |
Regulation of T cell cytokine expression by Tet2-mediated DNA demethylation |
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| Relations |
| BioSample |
SAMN03372256 |
| SRA |
SRX889919 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1618374_5mC.Th17.wig.gz |
717.8 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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