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| Status |
Public on May 31, 2013 |
| Title |
expanded FOXP3+ H3K27me3 |
| Sample type |
SRA |
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| Source name |
CD4+CD25highCD127low/- Treg cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type origin: Peripheral blood mononuclear cells (PBMC)
cell type: CD4+CD25highCD127low/- Treg
cell subtype: expanded human FOXP3+ Treg cells
days of expansion: 35
foxp3 status: positive
chip antibody: H3K27me3
chip antibody vendor: abcam
chip antibody cat. #: ab6002
chip antibody lot #: gr74123-1
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| Treatment protocol |
Expanded cells were sorted into FOXP3+ and FOXP3-losing cells by FACS (BD Biosciences, USA). For intracellular staining, washing and following cell sorting steps, the PBS with 0.1% diethyl pyrocarbonate (DEPC; Sigma-Aldrich, USA) was used. The buffer was autoclaved and supplemented with recombinant RNasin® Ribonuclease Inhibitor (Promega, USA) just before use.
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| Growth protocol |
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from leukapheresis products of healthy volunteers. CD4+ T cells were enriched with the human CD4+ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25highCD127low/- Treg cells were isolated by FACS (BD Biosciences, USA) from enriched CD4+ T cells and cultured in X-Vivo 15 medium (Lonza, USA) supplemented with 5% human AB serum (Sigma-Aldrich, USA). Cells were stimulated with anti-CD3/CD28 Abs-coated beads (Invitrogen-Dynal, USA) in the presence of 500 U/ml recombinant human IL-2 (PeproTech, USA) for 8 days and then rested in the presence of 100 U/ml IL-2 for 14 days. After two cycles of stimulation, cells were rested for 4-5 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde and chromatin was sonicated to obtain an average fragment length of 200 to 500 bp. After preclearing with protein A agarose beads (Upstate, USA), sonicated chromatin was precipitated with anti-H3K4m3 and anti-H3K27m3 (abcam, United Kingdom) overnight at 4℃. The immune complexes were bound to protein A agarose beads (Upstate, USA). After washing and elution, crosslinks were reversed at 65℃ overnight. The eluted DNA was treated sequentially with Proteinase K and RNase A, and purified with the QIAquick PCR-purification kit (Qiagen, Germany). The enrichment efficiency of ChIP was detected using qPCR approach.
The purified DNA fragments was repaired using PNK and Klenow enzyme, and ligated to adapters. Subsequently, PCR-amplified fragments of around 100-300bp were sequenced using Illumina HiSeq™ 2000 following manufacturer’s protocols (www.illumina.com).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample 2
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| Data processing |
Base-calling was performed using RTA and the data was transformed with OLB version 1.9.0.
Raw data were filtered using the following specifications. The adaptor sequence and low-quality sequence were removed. The low-quality read was defined as sequence whose Q20 ≥50%, or that contains more than 10% unkown base N.
Clean reads were mapped to the hg19 using SOAP version 2.21 with parameters -p 4 -l 23 -v 2 -s 35. Only alignments with ≤2 mismatching bases were retained.
Peaks were identified using MACS version 1.4.0 with default parameters.
Genome_build: UCSC human genome (hg19)
Supplementary_files_format_and_content: wig file
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| Submission date |
May 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Haiqi He |
| Organization name |
Xi'an Jiaotong University
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| Street address |
West Yanta Road
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| City |
Xi'an |
| State/province |
Shaanxi |
| ZIP/Postal code |
710061 |
| Country |
China |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE47510 |
Genome-wide maps of H3K4me3 and H3K27me3 in human FOXP3+ Treg cells and the corresponding FOXP3-losing cells |
| GSE47747 |
Contribution of histone methylation to the plasticity of human FOXP3+ Treg cells |
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| Relations |
| BioSample |
SAMN02183408 |
| SRA |
SRX288054 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1151310_FOXP3+_H3K27me3.bed.194_MACS.wig.gz |
102.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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