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| Status |
Public on Jun 06, 2013 |
| Title |
expanded FOXP3-losing |
| Sample type |
SRA |
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| Source name |
CD4+CD25highCD127low/- Treg cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD4+CD25highCD127low/- Treg cells
cell sorted: In vitro expanded FOXP3-losing Treg cells
time in culture: 35 days
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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 |
total RNA |
| Extraction protocol |
Total RNA was extacted using TriPure Reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Base-calling was performed using RTA version 1.12.4.2. and the data was transformed with BclConverter version 1.9.0.
Raw sequences were transformed into clean tags after removal of adaptor sequence, low-quality tags, tags with a copy number of 1 and tags that are too long or too short. The low-quality tags was defined as tags containing unkown base N.
The clean tags were mapped to hg19 using bowtie with parameters -f -l 21 -n 1 -k 2 -m 2 --un unmapped_tag . Clean tags mapped to reference sequences from multiple genes were filtered. Only tags with perfect matching or 1 bp mismatch were considered for further analysis.
The number of unambiguous clean tags for each gene was calculated and normalized to TPM (number of transcripts per million clean tags) using a protocol from Morrissy et al., Genome Res, 2009. The number of normalized sequence tags of a gene locus=( the number of mapped tags in the gene locus/ the total number of sequence tags of the whole genome)×1,000,000.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include gene symbols, TPM values, ontology information of gene-corresponding GO terms and so on.
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| Submission date |
Jun 04, 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) |
| GSE47636 |
Transcriptional profiling of FOXP3+ Treg cells and corresponding FOXP3-losing cells |
| GSE47747 |
Contribution of histone methylation to the plasticity of human FOXP3+ Treg cells |
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| Relations |
| BioSample |
SAMN02191479 |
| SRA |
SRX294979 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1153917_FOXP3-_Treg.GeneExpression.txt.gz |
922.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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