|
| Status |
Public on Feb 09, 2011 |
| Title |
WTTh17STAT3 (ChIP-Seq) |
| Sample type |
SRA |
| |
|
| Source name |
primary CD4+ T cells from spleen and lymph nodes
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: wild type
cell type: in vitro polarized T helper17 cells
passages: FACS sorted naive CD4+T cells cultured in vitro for 3 days under Th17 condition and restimulated with IL-6 for 30 minutes
chip antibody: 5 ug of STAT3 (14-6727: eBioScience) per IP
|
| Treatment protocol |
After 3 day culture in vitro, cells were restimulated for either IL-6 (40 ng/ml) or IL-2 (200 IU/ml) before chemically cross linked by 1% formaldehyde.
|
| Growth protocol |
Naïve CD4+CD44-CD62L+ T cells were isolated and sorted on the flow cytometer. Cells were cultured for 72h with platebound anti-CD3, anti-CD28 (10 mg/ml each), IL-6 (20 ng/ml), TGF-beta (2 ng/ml), anti-IL-4 (10 mg/ml) and anti-IL-2 (100IU/ml) for 3 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lysates were made by sonication and protein-DNA complexes were isolated with anti-STAT5 (STAT5a (PA-ST5A: R&D) and STAT5b (PA-ST5B: R&D) antibodies, 2 ul each per IP) and anti-STAT3 (14-6727: eBioScience, 5 ug per IP. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt, phosphorylated ends were treated with Taq polymerase and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP against STAT3
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse mm9 genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained. Cisgenome was used for peak-calling.
CisGenome ( Nature Biotechnology, 26: 1293-1300)
|
| |
|
| Submission date |
Jan 11, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Golnaz Vahedi |
| Organization name |
National Institutes of Health
|
| Department |
NIAMS
|
| Lab |
Lymphocyte Cell Biology Section
|
| Street address |
9000 Rockville Pike Bldg 10 Rm 13C101A
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL9185 |
| Series (2) |
| GSE26552 |
Genome-wide binding of STAT3 and STAT5 under Th17 conditions (ChIP-Seq) |
| GSE26553 |
Opposing regulation of the Il17 locus through direct, reciprocal actions of STAT3 and STAT5 |
|
| Relations |
| SRA |
SRX037909 |
| BioSample |
SAMN00190762 |
| Named Annotation |
GSM652877_Stat3Il6.bed.gz |
