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Sample GSM1618376 Query DataSets for GSM1618376
Status Public on Apr 12, 2015
Title 5hmC.Naive
Sample type SRA
Source name CD4+ T cells, 5hmC.Naive
Organism Mus musculus
Characteristics strain: C57BL/6
cell population: CD4+ T cells
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.
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.
Library strategy MeDIP-Seq
Library source genomic
Library selection 5-methylcytidine antibody
Instrument model Illumina HiSeq 2500
Description Reads obtained from two instruments: Illumina Genome Analyzer IIx and Illumina HiSeq 2500
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.
Submission date Feb 24, 2015
Last update date May 15, 2019
Contact name Raymond Doty
Organization name University of Washington
Department Division of Hematology
Street address Box 357710
City Seattle
State/province WA
ZIP/Postal code 98195
Country USA
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
BioSample SAMN03372251
SRA SRX889921

Supplementary file Size Download File type/resource
GSM1618376_5hmC.Naive.wig.gz 1.7 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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